Method for assessment of potential for development of dravet syndrome and use thereof

ABSTRACT

Provided is a method of assessing a potential for development of Dravet syndrome with high accuracy, and use thereof. The method according to the present invention of assessing a potential for development of Dravet syndrome includes, with use of a sample taken from a subject, detecting whether or not a mutation is on α-subunit type 1 of voltage-gated sodium ion channel Na V 1.1, and detecting whether or not a mutation is on α-subunit type 1 of voltage-gated calcium ion channel Ca V 2.1.

TECHNICAL FIELD

The present invention relates to a method for assessing a potential fordevelopment of Dravet syndrome, and use thereof.

BACKGROUND ART

Febrile seizure is a disease that has a high incidence rate ofapproximately 8% in infants. A main symptom of febrile seizure is knownas a continuation of generalized convulsions for 1 to 5 minutes whilesuffering a fever at or over 38° C. caused by a viral or bacterialinfection such as a cold, or microbism. Most cases of febrile seizurethat have an onset of between 6 months after birth and around 5 yearsold cure by the time when the patient turns 6 years old. In many cases,febrile seizure does not require active treatment. Therefore, febrileseizure is considered, in principle, as a benign disease.

However, among patients whose onset of febrile seizure was under the ageof one, other than the patients of the benign disease which cease as aregular febrile seizure, there are some patients who suffer fromconvulsions continuously even after turning 6 years old, and there aresome patients who are patients of Dravet syndrome (previously called“Severe Myoclonic Epilepsy in Infancy; SMEI”), which are patients of anintractable epilepsy disease.

The patients of Dravet syndrome are triggered in the onset ofconvulsions under the age of one. An average age of the onset ofconvulsions for patients of Dravet syndrome is 4 months to 6 monthsafter birth. An incipient seizure of convulsion for a patient of Dravetsyndrome is generally a systemic or a unilateral tonic-clonic or clonicconvulsion, and during infancy, may lead to status epilepticus.Moreover, this convulsion seizure is easily induced by fever or bathing.

Conventionally, febrile seizure was diagnosed and treated by a generalpediatrician or a family doctor, and Dravet syndrome is also diagnosedbased on clinical symptoms characteristic of Dravet syndrome such asconvulsion seizure or the like. However, by the time the patients ofDravet syndrome turn two to three years old, that is around when theclinical symptoms of Dravet syndrome have all appeared, these patientswould have suffered repetitive convulsions many times and would oftenhave had experienced critical conditions such as status epilepticus orthe like. Hence, it is necessary to develop a diagnosis method thatenables detection of Dravet syndrome in its possible earliest stage by ageneral pediatrician or family doctor, who is engaged in primary medicalcare. Detection of Dravet syndrome at an earlier stage would allow forthe patent to see an epilepsy specialist in advance, which would allowfor preventing the patient from reaching a critical condition.

Recently, it has been reported that 30% to 80% of Dravet syndromepatients find missense mutation (mutation causing a substitution of anamino acid) and nonsense mutation (mutation causing protein synthesis tostop in an incomplete state) on a SCN1A gene that encodes avoltage-gated sodium ion channel Na_(V)1.1 α-subunit type 1 (see NonPatent Literature 1 and 2). From such a point in view, attempts havebeen made to examine abnormalities in the SCN1A gene to diagnose Dravetsyndrome on the basis of genes.

For example, Patent Literatures 1 to 4 disclose that mutation of theSCN1A gene is related to SMEI. Moreover, Patent Literatures 1 to 4disclose that SMEI can be diagnosed by use of the mutation of the SCN1Agene as an indicator.

More specifically, Patent Literature 1 discloses the diagnosis of SMEIby assessing a plurality of mutations on the SCN1A gene that relate toSMEI, as a whole.

Patent Literature 2 discloses the diagnosis of SMEI performed bydetecting a presence of a mutation that frequently occurs on the SCN1Agene of a nerve that is affected by SMEI.

Patent Literatures 3 and 4 disclose a method of diagnosing epilepsysyndromes including SMEI and syndromes associated with SMEI, bydetecting a change in the SCN1A gene and confirming whether that changeis known as being related to SMEI or a syndrome associated with SMEI oris known as not being related to SMEI or a syndrome associated withSMEI.

CITATION LIST Patent Literature Patent Literature 1

-   Japanese Patent Application Publication, Tokukai, No. 2004-329153 A    (Publication Date: Nov. 25, 2004)

Patent Literature 2

-   Japanese Patent Application Publication, Tokukai, No. 2004-73058 A    (Publication Date: Mar. 11, 2004)

Patent Literature 3

-   Published Japanese Translations of PCT International Publication,    Tokuhyo, No. 2008-546376 A (Publication Date: Dec. 25, 2008)

Patent Literature 4

-   Published Japanese Translations of PCT International Publication,    Tokuhyo, No. 2006-524490 A (Publication Date: Nov. 2, 2006)

Non Patent Literature Non Patent Literature 1

-   Sugawara T, Mazaki-Miyazaki E, Fukushima K, Shimomura J, Fujiwara T,    Hamano S, Inoue Y, Yamakawa K. 2002. Frequent mutations of SCN1A in    severe myoclonic epilepsy in infancy. Neurology 58: 1122-1124.

Non Patent Literature 2

-   Ohmori I, Ouchida M, Ohtsuka Y, Oka E, Shimizu K. 2002. Significant    correlation of the SCN1A mutations and severe myoclonic epilepsy in    infancy. Biochem Biophys Res Commun 295: 17-23.

Non Patent Literature 3

-   Escayg A, Heils A, MacDonald B T, Haug K, Sander T, and Meisler    M H. 2001. A novel SCN1A mutation associated with generalized    epilepsy with febrile seizures plus—and prevalence of variants in    patients with epilepsy. Am J Hum Genet. 68: 866-873.

SUMMARY OF INVENTION Technical Problem

As described above, the mutation on the SCN1A gene is found in anextremely large number of Dravet syndrome patients (30% to 80%).However, it is becoming revealed that the presence of a mutation on theSCN1A gene does not necessarily mean that the symptoms of Dravetsyndrome would appear.

For example, Non Patent Literature 3 reports that not just the patientsof the intractable Dravet syndrome, but also patients of febrile seizureand patients with a certain kind of benign epilepsy (e.g. GEFS+(Generalized epilepsy with febrile seizure plus)) have a mutation on theSCN1A gene.

As such, the mutation on the SCN1A gene is not a phenomenon specific toDravet syndrome. Hence, the conventional methods of examining just theabnormalities on the SCN1A gene as described in Patent Literatures 1 to4 can be said as insufficient for specifically diagnosing Dravetsyndrome.

Therefore, in order to distinguish between the patients with benignfebrile seizure and the patients with Dravet syndrome and to allow forthe patients with Dravet syndrome to receive appropriate treatment by aspecialist, further development is required in techniques for moreaccurately diagnosing Dravet syndrome.

The present invention is accomplished in view of the foregoing problems,and an object thereof is to provide a method of (specifically) assessingwith high accuracy a potential for development of Dravet syndrome.

Solution to Problem

Patients of GEFS+ and the patients of Dravet syndrome are common in apoint that the SCN1A gene has a mutation. Meanwhile, the inventorsperformed diligent study based on their unique point of view of focusingon the difference in malignancy between the diseases; they consideredthat the development of Dravet syndrome is related to not just themutation on the SCN1A gene but also another factor, and that anothercause is related to the worsening and intractableness of Dravetsyndrome. As a result, the inventors uniquely found out that many Dravetsyndrome patients have a mutation on the SCN1A gene and further amutation on the CACNA1A gene that encodes a P/Q type voltage-gatedcalcium ion channel Ca_(V)2.1 α1 subunit.

Furthermore, based on this finding, the inventors produced a rat havingboth the mutations on the SCN1A gene and the CACNA1A gene, anddemonstrated that the rat having both the mutations on the SCN1A geneand the CACNA1A gene experienced more serious convulsion seizures ascompared to rats having just the mutation on the SCN1A gene.

Based on these results of analyzing genes and animal testing results, itwas found that the potential for development of Dravet syndrome can beassessed with high accuracy by detecting mutations for both α-subunittype 1 of voltage-gated sodium ion channel Na_(V)1.1 and α-subunit type1 of voltage-gated calcium ion channel Ca_(V)2.1, and accomplished thepresent invention.

Namely, the present invention includes the following inventions.

An assessment method according to the present invention is a method ofassessing a potential for development of Dravet syndrome, the methodincluding:

with use of a sample taken from a subject,

detecting whether or not a mutation is on α-subunit type 1 ofvoltage-gated sodium ion channel Na_(V)1.1; and

detecting whether or not a mutation is on α-subunit type 1 ofvoltage-gated calcium ion channel Ca_(V)2.1. It is preferable that theassessment method according to the present invention is a method ofobtaining data for assessing potential for development of Dravetsyndrome.

A kit according to the present invention is a kit for assessing apotential for development of Dravet syndrome, the kit comprising:

a polynucleotide being used for determining a mutation on α-subunit type1 of voltage-gated sodium ion channel Na_(V)1.1; and

a polynucleotide being used for determining a mutation on α-subunit type1 of voltage-gated calcium ion channel Ca_(V)2.1. The kit according tothe present invention may be a kit for obtaining data for assessing apotential for development of Dravet syndrome.

A model animal of Dravet syndrome according to the present invention hasa mutation on both α-subunit type 1 of voltage-gated sodium ion channelNa_(V)1.1 and α-subunit type 1 of voltage-gated calcium ion channelCa_(V)2.1.

A production method according to the present invention of a model animalof Dravet syndrome is a method of producing the model animal of Dravetsyndrome described above, which method includes:

introducing a mutation on a α-subunit type 1 of the voltage-gated sodiumion channel Na_(V)1.1; and

introducing a mutation on a α-subunit type 1 of the voltage-gatedcalcium ion channel Ca_(V)2.1.

A cell according to the present invention has a mutation on bothα-subunit type 1 of voltage-gated sodium ion channel Na_(V)1.1 andα-subunit type 1 of voltage-gated calcium ion channel Ca_(V)2.1.

A method of producing a cell according to the present invention is amethod of producing the cell described above, which method includes:

introducing a mutation on a α-subunit type 1 of the voltage-gated sodiumion channel Na_(V)1.1; and

introducing a mutation on a α-subunit type 1 of the voltage-gatedcalcium ion channel Ca_(V)2.1.

A screening method according to the present invention of a drug fortreating Dravet syndrome includes:

administering a candidate agent to the model animal of Dravet syndromeaccording to the present invention; and

assessing whether or not the administering of the candidate agent hasmade Dravet syndrome improve or cure in the model animal of Dravetsyndrome.

A screening method according to the present invention of a drug fortreating Dravet syndrome includes:

administering a candidate agent to the cell according to the presentinvention; and

assessing whether or not the administering of the candidate agent hasmade activity of the voltage-gated sodium ion channel Na_(V)1.1 and/oractivity of the voltage-gated calcium ion channel Ca_(V)2.1 change inthe cell.

For a fuller understanding of the nature and advantages of theinvention, reference should be made to the ensuing detailed descriptiontaken in conjunction with the accompanying drawings.

Advantageous Effects of Invention

The method according to the present invention of assessing a potentialfor development of Dravet syndrome allows for obtaining data forassessing the potential for development of Dravet syndrome, by detectingmutations for both α-subunit type 1 of voltage-gated sodium ion channel

Na_(V)1.1 and α-subunit type 1 of voltage-gated calcium ion channelCa_(V)2.1.

Patients of GEFS+, being a benign epilepsy, inherit the mutation of theSCN1A gene within the family. In comparison, in patients of Dravetsyndrome, approximately 90% of the mutations on SCN1A gene are de novomutation, i.e. are anew mutations in which a mutation arises even thoughtheir parents have no mutation. As such, although the GEFS+ patients andthe Dravet syndrome patients are common in that a mutation is on theSCN1A gene, the cause for the difference in malignancy of the diseasewas unknown. However, it was clarified by the present inventors for thefirst time, that the presence of mutations on both the SCN1A gene andthe CACNA1A gene is related to the worsening and intractableness ofDravet syndrome.

As described above, reports have already been made that a mutation onα-subunit type 1 of voltage-gated sodium ion channel Na_(V)1.1(hereinafter, referred to as “sodium ion channel α1 subunit”) is relatedto the development of Dravet syndrome. However, no reports have beenmade whatsoever that Dravet syndrome is related to a mutation onα-subunit type 1 of voltage-gated calcium ion channel Ca_(V)2.1(hereinafter, referred to as “calcium ion channel α1 subunit”).

Reports have been made that a mutation on a subunit other than the α 1subunit of voltage-gated calcium ion channel Ca_(V)2.1 is associatedwith Dravet syndrome (see Iori Ohmori et. Al., Neurobiology of Disease32 (2008) 349-354). More specifically, this literature (Iori Ohmori et.Al.) discloses that a mutation on β4 subunit of voltage-gated calciumion channel Ca_(V)2.1 (hereinafter, simply referred to as “calcium ionchannel (34 subunit”) is associated with Dravet syndrome.

However, the foregoing literature strongly teaches regarding Dravetsyndrome that a mutation on the “calcium ion channel β4 subunit” isimportant together with the mutation on the “α-subunit of sodium ionchannel Na_(V)1.1”. This description in the literature hinders amotivation to arrive at a point that a mutation suitable for detectingDravet syndrome is present in the calcium ion channel α 1 subunit.

In the first place, a skilled person would not arrive at considering,just because a relationship of a mutation on a specific subunit with adisease is known for a specific channel, that other subunits would alsohave a mutation related to that disease. At least, the finding that thevoltage-gated sodium ion channel Na_(V)1.1 is related to Dravet syndromeis only known regarding the mutation on the “α 1 subunit”; this does notgive motivation for analyzing mutations on other subunits.

As to a mutation on the calcium ion channel α 1 subunit, reports havebeen made stating a relationship with (1) epixodic ataxia type 2(characterized in paroxysmal cerebellar ataxia), (2) familial hemiplegicmigraine type 1 (e.g. hemiplegia, hemianopsia, dysphagia, throbbingheadache), and (3) spinocerebellar ataxia type 6 (e.g. ataxic gait, limbataxia, cerebellar dysarthria, nystagmus) (see Keiji IMOTO et al.,“Igaku no Ayumi” (Development in Medical Science), Vol. 201, No. 13(Issued Jun. 29, 2002); Taiji TSUNEMI et al., “Igaku no Ayumi”(Development in Medical Science), Vol. 201, No. 13 (Issued Jun. 29,2002)). However, the diseases of (1) to (3) all show no symptoms ofepilepsy, and neither are diseases related to Dravet syndrome. At least,although the finding regarding the mutation on the calcium ion channel α1 subunit is known as related to the diseases of (1) to (3), it is notone that gives motivation for analyzing a mutation on the calcium ionchannel α 1 subunit in Dravet syndrome, which disease is completelyunrelated to the diseases of (1) to (3).

The assessment method according to the present invention detects amutation on α-subunit type 1 of the voltage-gated sodium ion channelNa_(V)1.1 and on α-subunit type 1 of the voltage-gated calcium ionchannel Ca_(V)2.1. Hence, it is possible to detect Dravet syndrome withhigh accuracy. Consequently, the assessment method of the presentinvention brings about an effect that it is possible to improvereliability of a potential for detecting Dravet syndrome as compared tothe conventional method by detecting a mutation on the SCN1A gene.Furthermore, detection of a mutation on α-subunit type 1 of thevoltage-gated sodium ion channel Na_(V)1.1 and a mutation on α-subunittype 1 of the voltage-gated calcium ion channel Ca_(V)2.1 is possibleeven with an infant under the age of one. Hence, according to theassessment method of the present invention, an effect is brought aboutthat data for assessing the potential for development in Dravet syndromecan be obtained from a patient in an early stage of development or in astage prior to the onset of the intractable disease, in particular of aninfant under the age of one.

Moreover, as shown in Examples later described, an effect is broughtabout that by detecting a mutation on both α-subunit type 1 of thevoltage-gated sodium ion channel Na_(V)1.1 and α-subunit type 1 of thevoltage-gated calcium ion channel Ca_(V)2.1, the detection sensitivityof Dravet syndrome patients dramatically improve.

Furthermore, with use of the kit according to the present invention, itis possible to easily detect the mutation on both α-subunit type 1 ofthe voltage-gated sodium ion channel Na_(V)1.1 and α-subunit type 1 ofthe voltage-gated calcium ion channel Ca_(V)2.1. Hence, the kitaccording to the present invention is useful for a general pediatricianto screen, at an early stage of disease of under the age of one, apatient of Dravet syndrome that requires treatment by a specialist,among benign febrile epilepsy.

By using the assessment method and kit according to the presentinvention, it is possible to detect the patients of Dravet syndrome withhigh accuracy at the point in time of an age under one, which is an agedifficult to detect until now. Moreover, by sending a blood sample to anexamination center and examining its abnormal genes, it is possible todetect a Dravet syndrome patient with high accuracy even in a privatehospital at a remote location or the like.

Moreover, the Dravet syndrome model animal and cell according to thepresent invention can be usefully used for resolving a developmentmechanism of the intractable Dravet syndrome, and for development andthe like of medicament for Dravet syndrome.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a view illustrating an amino acid sequence of a proteinencoded by a human SCN1A gene and an amino acid sequence of a proteinencoded by a rat Scn1a gene.

FIG. 2 is a view illustrating a result of performing function analysisof sodium ion channel, by use of patch clamping. Illustrated in (a) is atypical example of a sodium current effected by a change in potential ofa normal sodium ion channel and a mutant sodium ion channel. Illustratedin (b) is a result of examining a time constant (τ) at inactivation.

FIG. 3 is a view illustrating a result of performing function analysisof a sodium ion channel, by use of patch clamping. Illustrated in (a) isa current-voltage relationship, illustrated in (b) is an activationcurve of the sodium ion channel, illustrated in (c) is an inactivationcurve of the sodium ion channel, and illustrated in (d) is a recoverycurve from the inactivation of the sodium ion channel.

FIG. 4 is a view illustrating a result of performing function analysisof a sodium ion channel, by use of patch clamping. Illustrated in (a) isa sodium current flowing in the sodium ion channel, and illustrated in(b) is a relative value (%) of a persistent sodium current amountflowing into the sodium ion channel.

FIG. 5 is a view illustrating genotypes of parent rats (P), first filialgeneration (F1) rats, and second filial generation (F2) rats.Illustrated in (a) is a view showing genotypes of the parent rats (P)and the F1 rats. Illustrated in (b) are genotypes of the F1 rats and theF2 rats.

FIG. 6 is a view illustrating a method of identifying genotypes of theScn1a gene and the Cacna1a gene of the F2 rat, by sequencing.

FIG. 7 is a view illustrating a method of identifying a genotype of theScn1a gene of the F2 rat, by restriction enzyme digestion. Illustratedin (a) is a nucleotide sequence of where mutation is on a mutant Scn1agene (N1417H), and a nucleotide sequence of a wild-type Scn1a genecorresponding to that nucleotide sequence of the mutant Scn1a gene.Illustrated in (b) is a size of a DNA fragment expected by therestriction enzyme digestion. Illustrated in (c) is a result ofelectrophoresis.

FIG. 8 is a view illustrating a method of identifying a genotype of theCacna1a gene in a F2 rat, by restriction enzyme digestion. Illustratedin (a) is a nucleotide sequence of where a mutation is on a mutantCacna1a gene (M251K), and a nucleotide sequence of a wild-type Cacna1agene corresponding to that nucleotide sequence of the mutant Cacna1agene. Illustrated in (b) is a size of a DNA fragment expected by therestriction enzyme digestion. Illustrated in (c) is a result ofelectrophoresis.

FIG. 9 is a view illustrating a result of examining an effect of amutation on the Cacna1a gene, in a rat having a mutation on Scn1a gene.Illustrated in (a) is a body temperature at a time of convulsion onset(convulsion threshold), illustrated in (b) is a severity score, andillustrated in (c) is duration of the convulsion.

FIG. 10 is a view illustrating a part of an electroencephalogram at atime of seizure of a rat in group (3) (Scn1a mutant (homo)+Cacna1amutant (hetero)).

FIG. 11 is a view illustrating an amino acid sequence of a proteinencoded by a human CACNA1A gene and an amino acid sequence of a proteinencoded by a rat Cacna1a gene.

FIG. 12 is a view illustrating a result of detecting a mutation onvoltage-gated calcium ion channel Ca_(V)2.1 a 1 subunit. Illustrated in(a) is a result of a mutation analysis of the CACNA1A gene, andschematically illustrated in (b) is a part where a mutation was detectedin the calcium ion channel α1 subunit.

FIG. 13 is a view illustrating a result of performing function analysisof the calcium ion channel, by use of patch clamping. Illustrated in (a)is a barium current record effected by a change in potential of a normalcalcium ion channel and a mutant calcium ion channel. Illustrated in (b)is a current-voltage relationship, and illustrated in (c) is peakcurrent value (pA), a total charge (pF) and a peak current density(pA/pF).

FIG. 14 is a view illustrating a result of performing function analysisof a calcium ion channel, by use of patch clamping. Illustrated in (a)is an activation curve of the calcium ion channel. Illustrated in (b) isa time constant of voltage-gated activation of the calcium ion channel.Illustrated in (c) is a time constant of voltage-gated activation at 20mV. Illustrated in (d) is a voltage-gated inactivation curve of thecalcium ion channel. Illustrated in (e) is a result of examining fastand slow inactivation time constants (τ).

DESCRIPTION OF EMBODIMENTS

Described below is an embodiment of the present invention in detail. Thepresent invention is not limited to this embodiment however, and may becarried out in modes of various modifications that are made within thedescribed scope. Moreover, all academic literature and patent literaturedisclosed in the present specification are incorporated as reference.Unless mentioned otherwise, numerical ranges expressed as “A to B”denote “not less than A but not more than B”.

1. Assessment method according to the present invention

A method of assessing a potential for development of Dravet syndromeaccording to the present invention (also referred to as “assessmentmethod according to the present invention”) is a method of assessing apotential for development of Dravet syndrome in a subject, by use of asample taken from the subject. In the present specification, the“potential for development of Dravet syndrome” includes a potential thatthe Dravet syndrome is already developed and a potential that the Dravetsyndrome may develop in the future.

The subject is not particularly limited, and may be an individual inwhich Dravet syndrome has developed (individual having potential fordevelopment) or may be an individual in which the Dravet syndrome is notdeveloped (individual having no potential for development). Out of suchindividuals, it is preferable that the subject is of either infants orchildren.

The assessment method according to the present invention, morespecifically, may be of any method as long as it includes, with use of asample taken from the subject: detecting whether or not a mutation is onα-subunit type 1 of voltage-gated sodium ion channel Na_(V)1.1; anddetecting whether or not a mutation is on α-subunit type 1 ofvoltage-gated calcium ion channel Ca_(V)2.1. Any other specificconfigurations are not limited in particular.

In the embodiment, the voltage-gated sodium ion channel Na_(V)1.1 ismade up of α-subunit type 1, β₁ subunit, and β₂ subunit. The β₁ subunitand the β₂ subunit are auxiliary subunits.

The α-subunit type 1 of voltage-gated sodium ion channel Na_(V)1.1(hereinafter, referred to as “sodium ion channel α1 subunit”) is forexample a polypeptide that is registered as GenBank accession No.AB093548 (i.e. amino acid sequence represented by SEQ ID NO. 1).Moreover, an example of a gene that encodes the α-subunit type 1 ofvoltage-gated sodium ion channel Na_(V)1.1 (hereinafter, called “sodiumion channel α1 subunit gene”) is, as a SCN1A gene, a polynucleotide madeup of a nucleotide sequence registered as GenBank accession No. AB093548(i.e. nucleotide sequence represented by SEQ ID NO. 2).

The voltage-gated calcium ion channel Ca_(V)2.1 is made up of α-subunittype 1, β subunit, γ subunit, and α2δ subunit.

The voltage-gated calcium ion channel Ca_(V)2.1 α-subunit type 1(hereinafter, referred to as “calcium ion channel al subunit”) is forexample a polypeptide registered as GenBank accession No. NM 023035(i.e. amino acid sequence represented by SEQ ID NO. 3). Moreover, anexample of a gene that codes the α-subunit type 1 of voltage-gatedcalcium ion channel Ca_(V)2.1 (hereinafter, referred to as “calcium ionchannel α1 subunit gene”) is, as a CACNA1A gene, a polynucleotide madeup of a nucleotide sequence registered as GenBank accession No. NM023035 (i.e. nucleotide sequence represented by SEQ ID NO. 4).

In the present specification, for example, the term “α-subunit type 1 ofvoltage-gated sodium ion channel Na_(V)1.1” denotes “α-subunit type 1protein of voltage-gated sodium ion channel Na_(V)1.1”. Namely, in thepresent specification, unless it is clearly described as indicating agene as like “gene encoding α-subunit type 1 of voltage-gated sodium ionchannel Na_(V)1.1” or “α-subunit type 1 gene of voltage-gated sodium ionchannel Na_(V)1.1”, a protein is denoted. This way of description is notlimited to the “α-subunit type 1 of voltage-gated sodium ion channelNa_(V)1.1”, and “α-subunit type 1 of voltage-gated calcium ion channelCa_(V)2.1” is denoted similarly thereto.

It is preferable that the assessment method according to the presentinvention further includes, in addition to the detecting the presence ofa mutation: detecting a change in activity of the voltage-gated sodiumion channel Na_(V)1.1; and detecting a change in activity of thevoltage-gated calcium ion channel Ca_(V)2.1.

The assessment method according to the present invention may include,for detecting the mutation, a step such as preprocessing of a samplethat is taken from the living organism. The “preprocessing” indicates,for example, a process of extracting DNA from the sample taken from theliving organism, a process of extracting RNA from the sample taken fromthe living organism, a process of extracting protein from the sampletaken from the living organism, or like process. These preprocessing canbe carried out by use of conventionally known methods.

The assessment method according to the present invention may be a methodof obtaining data for assessing a potential for development of Dravetsyndrome. In this case, the present invention does not include the stepof determining by a doctor.

(1-1. Detecting Presence of Mutation)

In the present specification, the “detecting presence of a mutation”denotes detecting a presence of a mutation on α-subunit type 1 ofvoltage-gated sodium ion channel Na_(V)1.1 and detecting a presence of amutation on α-subunit type 1 of voltage-gated calcium ion channelCa_(V)2.1.

In the assessment method according to the present invention, thedetecting of the presence of a mutation on the α-subunit type 1 ofvoltage-gated sodium ion channel Na_(V)1.1 may be performed prior to thedetecting of the presence of a mutation on the α-subunit type 1 ofvoltage-gated calcium ion channel Ca_(V)2.1 or vice versa, or may beperformed simultaneously.

By detecting the presence of a mutation in both the sodium ion channelα1 subunit and the calcium ion channel α1 subunit, it is possible toobtain the data that enables accurate assessment of the potential fordevelopment of Dravet syndrome.

The mutation detected by the assessment method according to the presentinvention may be a mutation on a nucleotide sequence of a gene, or maybe a mutation on an amino acid of a protein. The “mutation on anucleotide sequence of a gene” is not limited in particular by aspecific kind of mutation as long as it is a mutation that causes achange in an amino acid sequence of a protein encoded by a gene having amutation on its nucleotide sequence as compared to an amino acidsequence of a protein encoded by a wild-type gene. Mutations on thenucleotide sequence as described above are, for example, missensemutation (substitution of an amino acid), nonsense mutation (synthesisof an amino acid stops in an incomplete state), frameshift (a frame ofan amino acid codon shifts caused by insertion or deletion of anucleotide, which causes an amino acid sequence downstream of themutation position to change, thereby losing its original function),splicing defect (e.g. deletion of its exon region), minority nucleotideinsertion or deletion (a part of amino acids is newly added or losthowever its downstream is synthesized as normal amino acid), and minordeletion of an exon region (loss of one or a plurality of exon).Variations on the nucleotide sequence as such are not limited tomutations, and may also include gene polymorphism.

Moreover, in the assessment method according to the present invention,the detection of mutation may be performed to mRNA, cDNA, and proteinsobtained from these genes.

In the present specification, “gene” can be replaced by“polynucleotide”, “nucleic acid” or “nucleic acid molecule”.

The “polynucleotide” means a polymer of a nucleotide. Hence, the term“gene” in the present specification includes not only the doublestranded DNA but also a single stranded DNA and RNA (mRNA, etc.) such asa sense strand and an antisense strand that construct the doublestranded DNA.

The term “DNA” encompasses cDNA, genomic DNA and the like that can beobtained by cloning, a chemically synthesized technique or a combinationof these. Namely, DNA may be a “genome” type DNA, which includes anoncoding sequence such as intron or the like that is a form included inan animal genome, or may be a cDNA obtained from mRNA with use ofreverse transcriptase or polymerase, i.e. “transcription” type DNA thatdoes not include a noncoding sequence such as intron.

Examples of the mutation on sodium ion channel a 1 subunit is, morespecifically, a mutation of asparagine (N) at position 1417 of the aminoacid sequence of sodium ion channel α 1 subunit represented by SEQ IDNO. 1, and is preferably a mutation of asparagine (N) at position 1417to histidine (H) (“N1417H” in Table 1). This mutation is caused by, forexample, a mutation of adenine (A) at position 4249 of the nucleotidesequence of sodium ion channel α 1 subunit gene represented by SEQ IDNO. 2, preferably a substitution of adenine (A) at position 4249 withcytosine (C) (A4249C).

Moreover, another embodiment is a mutation of lysine (K) at position1027 of the amino acid sequence of the sodium ion channel α1 subunitrepresented by SEQ ID NO. 1, preferably a mutation of lysine (K) atposition 1027 to a stop codon (“K1027X” in Table 1). This mutation iscaused by, for example, a mutation of adenine (A) at position 3079 ofthe nucleotide sequence of sodium ion channel α1 subunit generepresented by SEQ ID NO. 2, preferably a substitution of adenine (A) atposition 3079 with thymine (T) (A3079T).

Yet another embodiment is a mutation of glutamine (Q) at position 1450of the amino acid sequence of sodium ion channel α1 subunit representedby SEQ ID NO. 1, preferably a mutation of glutamine (Q) at position 1450to arginine (R) (“Q1450R” in Table 1). This mutation is caused by, forexample, a mutation of adenine (A) at position 4349 of a nucleotidesequence of sodium ion channel α1 subunit gene represented by SEQ ID NO.2, preferably a substitution of adenine (A) at position 4349 withguanine (G) (A4349G).

Yet another embodiment is a mutation of threonine (T) at position 1082of the amino acid sequence of sodium ion channel α1 subunit representedby SEQ ID NO. 1, preferably a mutation causing generation of a stopcodon at position 1086 by frameshift (“T1082fsX1086” in Table 1). Thismutation is caused by, for example, a mutation of cytosine (C) atposition 3245 of a nucleotide sequence of sodium ion channel a 1 subunitgene represented by SEQ ID NO. 2, preferably a deletion of cytosine (C)at position 3245 (C3245de1).

Yet another embodiment is a mutation of lysine (K) at position 547 ofthe amino acid sequence of the sodium ion channel α1 subunit representedby SEQ ID NO. 1, preferably a mutation causing generation of a stopcodon at position 570 by frameshift (“K547fsX570” in Table 1). Thismutation is caused by, for example, a mutation at position 1641 of thenucleotide sequence of the sodium ion channel α1 subunit generepresented by SEQ ID NO. 2, preferably an insertion of adenine (A) intoposition 1641 (1641insA).

Yet another embodiment is a mutation of proline (P) at position 707 ofthe amino acid sequence of sodium ion channel α1 subunit represented bySEQ ID NO. 1, preferably a mutation causing generation of a stop codonat position 714 by frameshift (“P707fsX714” in Table 1). This mutationis caused by, for example, a mutation of cytosine (C) at position 2120in the nucleotide sequence of sodium ion channel al subunit generepresented by SEQ ID NO. 2, preferably a deletion of cytosine (C) atposition 2120 (C2120de1).

Yet another embodiment is a mutation of arginine (R) at position 712 ofthe amino acid sequence of sodium ion channel α1 subunit represented bySEQ ID NO. 1, preferably a mutation of arginine (R) at position 712 to astop codon (“R712X” in Table 1). This mutation is caused by, forexample, a mutation of cytosine (C) at position 2134 of the nucleotidesequence of the sodium ion channel α 1 subunit gene represented by SEQID NO. 2, preferably a substitution of cytosine (C) at position 2134with thymine (T) (C2134T).

Yet another embodiment is a mutation of leucine (L) at position 1265 ofthe amino acid sequence of the sodium ion channel α 1 subunitrepresented by SEQ ID NO. 1, preferably a mutation of leucine (L) atposition 1265 to proline (P) (“L1265P” in Table 1). This mutation iscaused by, for example, a mutation of thymine (T) at position 3794 ofthe nucleotide sequence of sodium ion channel α 1 subunit generepresented by SEQ ID NO. 2, preferably a substitution of thymine (T) atposition 3794 with cytosine (C) (T3794C).

Yet another embodiment is a deletion of amino acid of positions 460 to554 of the amino acid sequence of the sodium ion channel α 1 subunitrepresented by SEQ ID NO. 1 (“Exon10” in Table 1). This mutation iscaused by, for example, a deletion of nucleotide at positions 1378 to1662 (exon 10) of the nucleotide sequence of sodium ion channel α 1subunit gene represented by SEQ ID NO. 2.

Yet another embodiment is a mutation of arginine (R) at position 865 ofthe amino acid sequence of the sodium ion channel α 1 subunitrepresented by SEQ ID NO. 1, preferably a mutation of arginine (R) atposition 865 to a stop codon (“R865X” in Table 1). This mutation iscaused by, for example, a mutation of cytosine (C) at position 2593 ofthe nucleotide sequence of the sodium ion channel α 1 subunit generepresented by SEQ ID NO. 2, preferably a substitution of cytosine (C)at position 2593 with thymine (T) (C2593T).

Yet another embodiment is a mutation of arginine (R) at position 1648 ofthe amino acid sequence of sodium ion channel α 1 subunit represented bySEQ ID NO. 1, preferably a substitution of arginine (R) at position 1648with cysteine (C) (“R1648C” in Table 1). This mutation is caused by, forexample, a mutation of cytosine (C) at position 4942 of the nucleotidesequence of sodium ion channel α 1 subunit gene represented by SEQ IDNO. 2, preferably a substitution of cytosine (C) at position 4942 withthymine (T) (C4942T).

Yet another embodiment is a mutation of arginine (R) at position 931 inthe amino acid sequence of sodium ion channel α1 subunit represented bySEQ ID NO. 1, preferably a substitution of arginine (R) at position 931with cysteine (C) (“R931C” in Table 1). This mutation is caused by, forexample, a mutation of cytosine (C) at position 2791 of the nucleotidesequence of sodium ion channel α 1 subunit gene represented by SEQ IDNO. 2, preferably a substitution of cytosine (C) at position 2791 withthymine (T) (C2791T).

Yet another embodiment is a mutation of arginine (R) at position 501 inthe amino acid sequence of sodium ion channel α1 subunit represented bySEQ ID NO. 1, preferably a mutation causing generation of a stop codonat position 543 by frameshift (“R501fsX543” in Table 1). This mutationis caused by, for example, a mutation of guanine (G) at position 1502 ofthe nucleotide sequence of sodium ion channel al subunit generepresented by SEQ ID NO. 2, preferably a deletion of guanine (G) atposition 1502 (G1502de1).

Yet another embodiment is a mutation of alanine (A) at position 1002 inthe amino acid sequence of sodium ion channel α1 subunit represented bySEQ ID NO. 1, preferably a mutation causing generation of a stop codonat position 1009 by frameshift (“A1002fsX1009” in Table 1). Thismutation is caused by, for example, a mutation of cytosine (C) atposition 3006 of the nucleotide sequence of sodium ion channel alsubunit gene represented by SEQ ID NO. 2, preferably a deletion ofcytosine (C) at position 3006.

Yet another embodiment is a mutation of phenylalanine (F) at position902 of the amino acid sequence of sodium ion channel α1 subunitrepresented by SEQ ID NO. 1, preferably a mutation of phenylalanine (F)at position 902 to cysteine (C) (“F902C” in Table 1). This mutation iscaused by, for example, a mutation of thymine (T) at position 2705 ofthe nucleotide sequence of sodium ion channel α1 subunit generepresented by SEQ ID NO. 2, preferably by a substitution of thymine (T)at position 2705 with guanine (G) (T2705G).

Yet another embodiment is a mutation of glycine (G) at position 1674 ofthe amino acid sequence of aodium ion channel α1 subunit represented bySEQ ID NO. 1, preferably a substitution of glycine (G) at position 1674with arginine (R) (“G1674R” in Table 1). This mutation is caused by, forexample, a mutation of guanine (G) at position 5020 of the nucleotidesequence of sodium ion channel α1 subunit gene represented by SEQ ID NO.2, preferably a substitution of guanine (G) at position 5020 withcytosine (C) (G5020C).

Yet another embodiment is a mutation of valine (V) at position 1390 ofthe amino acid sequence of sodium ion channel α1 subunit represented bySEQ ID NO. 1, preferably a mutation of valine (V) at position 1390 tomethionine (M) (“V1390M” in Table 1). This mutation is caused by, forexample, a mutation of guanine (G) at position 4168 of the nucleotidesequence of sodium ion channel α1 subunit gene represented by SEQ ID NO.2, preferably a substitution of guanine (G) at position 4168 withadenine (A) (G4168A).

Yet another embodiment is a mutation of serine (S) at position 607 inthe amino acid sequence of sodium ion channel α1 subunit represented bySEQ ID NO. 1, preferably a mutation causing generation of a stop codonat position 622 by frameshift (“S607fsX622” in Table 1). This mutationis caused by, for example, a mutation of cytosine (C) at position 1820of the nucleotide sequence of sodium ion channel al subunit generepresented by SEQ ID NO. 2, preferably a deletion of cytosine (C) atposition 1820 (C1820de1).

Yet another embodiment is a mutation of tryptophan (W) at position 1434of the amino acid sequence of sodium ion channel α 1 subunit representedby SEQ ID NO. 1, preferably a substitution of tryptophan (W) at position1434 with arginine (R) (“W1434R” in Table 1). This mutation is caused bya mutation of thymine (T) at position 4300 of the nucleotide sequence ofsodium ion channel α 1 subunit gene represented by SEQ ID NO. 2,preferably a substitution of thymine (T) at position 4300 with cytosine(C) (T4300C).

Yet another embodiment is a mutation of threonine (T) at position 1909of the amino acid sequence of sodium ion channel α 1 subunit representedby SEQ ID NO. 1, preferably a substitution of threonine (T) at position1909 with isoleucine (I) (“T1909I” in Table 1). This mutation is causedby, for example, the mutation of cytosine (C) at position 5726 of thenucleotide sequence of sodium ion channel α 1 subunit gene representedby SEQ ID NO. 2, preferably by a substitution of cytosine (C) atposition 5726 with thymine (T) (C5726T).

Yet another embodiment is a mutation of phenylalanine (F) at position1289 of the amino acid sequence of sodium ion channel α 1 subunitrepresented by SEQ ID NO. 1, preferably a deletion of phenylalanine (F)at position 1289 (“F1289de1” in Table 1). This mutation is caused by,for example, mutations of cytosine (C) at position 3867, thymine (T) atposition 3868, and thymine (T) at position 3869, each in the nucleotidesequence of sodium ion channel α1 subunit gene represented by SEQ ID NO.2, preferably a deletion of cytosine (C) at position 3867, thymine (T)at position 3868, and thymine (T) at position 3869.

Yet another embodiment is a mutation of tryptophan (W) at position 1271of the amino acid sequence of sodium ion channel α1 subunit representedby SEQ ID NO. 1, preferably a mutation of tryptophan (W) at position1271 to a stop codon (“W1271X” in Table 1). This mutation is caused by,for example, a mutation of guanine (G) at position 3812 of thenucleotide sequence of sodium ion channel α1 subunit gene represented bySEQ ID NO. 2, preferably by a substitution of guanine (G) at position3812 with adenine (A) (G3812A).

Yet another embodiment is a mutation of alanine (A) at position 1429 ofthe amino acid sequence of sodium ion channel α1 subunit represented bySEQ ID NO. 1, preferably a mutation causing generation of a stop codonat position 1443 by frameshift (“A1429fsX1443” in Table 1). Thismutation is caused by, for example, a mutation of five-nucleotide CCACAbetween positions 4286 to 4290 of the nucleotide sequence of sodium ionchannel α 1 subunit gene represented by SEQ ID NO. 2, preferably asubstitution of CCACA at positions 4286 to 4290, with ATGTCC.

Moreover, another embodiment is a mutation of glycine (G) at position1880 of the amino acid sequence of sodium ion channel α1 subunitrepresented by SEQ ID NO. 1, preferably a mutation causing generation ofa stop codon at position 1881 by frameshift (“G1880fsX1881” in Table 1).This mutation is caused by mutation of six-nucleotide AGAGAT betweenpositions 5640 to 5645 of the nucleotide sequence of sodium ion channelα1 subunit gene represented by SEQ ID NO. 2, preferably a substitutionof six-nucleotide AGAGAT between positions 5640 to 5645 with CTAGAGTA.

Yet another embodiment is a mutation of alanine (A) at position 1685 ofthe amino acid sequence of sodium ion channel α1 subunit represented bySEQ ID NO. 1, preferably a substitution of alanine (A) at position 1685with aspartic acid (D) (“A1685D” in Table 1). This mutation is causedby, for example, a mutation of cytosine (C) at position 5054 of thenucleotide sequence of sodium ion channel α1 subunit gene represented bySEQ ID NO. 2, preferably by a substitution of cytosine (C) at position5054 with adenine (A) (C5054A).

Yet another embodiment is a mutation of arginine (R) at position 377 ofthe amino acid sequence of sodium ion channel α1 subunit represented bySEQ ID NO. 1, preferably a substitution of arginine (R) at position 377with leucine (L) (“R377L” in Table 1). This mutation is caused by, forexample, a mutation of guanine (G) at position 1130 of the nucleotidesequence of sodium ion channel α1 subunit gene represented by SEQ ID NO.2, preferably by substitution of guanine (G) at position 1130 withthymine (T) (G1130T).

Yet another embodiment is a mutation of serine (S) at position 1574 ofthe amino acid sequence of sodium ion channel α 1 subunit represented bySEQ ID NO. 1, preferably a mutation of serine (S) at position 1574 to astop codon (“51574X” in Table 1). This mutation is caused by, forexample, a mutation of cytosine (C) at position 4721 of the nucleotidesequence of sodium ion channel α 1 subunit gene represented by SEQ IDNO. 2, preferably a substitution of cytosine (C) at position 4721 withguanine (G) (C4721G).

Yet another embodiment is a mutation of glutamine (Q) at position 1277in the amino acid sequence of the sodium ion channel α 1 subunitrepresented by SEQ ID NO. 1, preferably a mutation of glutamine (Q) atposition 1277 to a stop codon (“Q1277X” in Table 1). This mutation iscaused by, for example, a mutation of cytosine (C) at position 3829 ofthe nucleotide sequence of sodium ion channel α 1 subunit generepresented by SEQ ID NO. 2, preferably by a substitution of cytosine(C) at position 3829 with thymine (T) (C3829T).

Yet another embodiment is a mutation of glycine (G) at position 177 ofthe amino acid sequence of sodium ion channel α1 subunit represented bySEQ ID NO. 1, preferably a mutation of glycine (G) at position 177 toarginine (R) (“G 177R” in Table 1). This mutation is caused by, forexample, a mutation of guanine (G) at position 529 of the nucleotidesequence of sodium ion channel α 1 subunit gene represented by SEQ IDNO. 2, preferably by a substitution of guanine (G) at position 529 withadenine (A) (G529A).

Yet another embodiment is a mutation of glutamic acid (E) at position788 of the amino acid sequence of sodium ion channel α 1 subunitrepresented by SEQ ID NO. 1, preferably a substitution of glutamic acid(E) at position 788 with lysine (K) (“E788K” in Table 1). This mutationis caused by, for example, a mutation of guanine (G) at position 2362 ofthe nucleotide sequence of sodium ion channel α 1 subunit generepresented by SEQ ID NO. 2, preferably by a substitution of guanine (G)at position 2362 with adenine (A) (G2362A).

Yet another embodiment is splicing defects at positions 1429 andsubsequent positions of the amino acid sequence of sodium ion channel α1subunit represented by SEQ ID NO. 1, preferably a deletion of positionsat and subsequent to 1429 (“intron 21” in Table 1). This mutation iscaused by, for example, a mutation of adenine (A) at a second lastposition (position −2), preferably a mutation in which adenine (A) at asecond last position (position −2) of the intron 21 is substituted withguanine (G) (intron 21 ag(−2)gg), out of the intron 21 present in agenomic DNA between positions 4284 and 4285 of the nucleotide sequenceof sodium ion channel a 1 subunit gene represented by SEQ ID NO. 2.Namely, the second last nucleotide sequence of the intron 21 present inthe genomic DNA between positions 4284 (exon 21) and 4285 (exon 22) ofthe nucleotide sequence of sodium ion channel a 1 subunit generepresented by SEQ ID NO. 2 is ag, and is connected to the beginning ofthe exon 22. Generally, since the ag of the intron 21 is a recognitionsequence that is spliced, in a case in which an abnormality exists atthat position, the intron is determined as still continuing, which thuscauses the exon immediately after (or in its downstream) to beabnormally spliced. This makes it impossible to generate a full-lengthprotein.

Yet another embodiment is a mutation of serine (S) at position 1574 ofthe amino acid sequence of sodium ion channel α 1 subunit represented bySEQ ID NO. 1, preferably a mutation of serine (S) at position 1574 to astop codon (“51574X” in Table 1). This mutation is caused by, forexample, a mutation of cytosine (C) at position 4721 of the nucleotidesequence of sodium ion channel α 1 subunit gene represented by SEQ IDNO. 2, preferably a substitution of cytosine (C) at position 4721 withguanine (G).

Yet another embodiment is a mutation of valine (V) at position 212 ofthe amino acid sequence of sodium ion channel α1 subunit represented bySEQ ID NO. 1, preferably a substitution of valine (V) at position 212with alanine (A) (“V212A” in Table 1). This mutation is caused by, forexample, a mutation of thymine (T) at position 635 of the nucleotidesequence of sodium ion channel α 1 subunit gene represented by SEQ IDNO. 2, preferably a substitution of thymine (T) at position 635 withcytosine (C) (T635C).

Yet another embodiment is a mutation of threonine (T) at position 1539of the amino acid sequence of sodium ion channel α 1 subunit representedby SEQ ID NO. 1, preferably a mutation of threonine (T) at position 1539to proline (P) (“T1539P” in Table 1). This mutation is caused by, forexample, a mutation of adenine (A) at position 4615 of the nucleotidesequence of sodium ion channel α 1 subunit gene represented by SEQ IDNO. 2, preferably a substitution of adenine (A) at position 4615 withcytosine (C) (A4615C).

Yet another embodiment is a mutation of tryptophan (W) at position 738of the amino acid sequence of sodium ion channel α1 subunit representedby SEQ ID NO. 1, preferably by mutation causing generation of a stopcodon at position 746 by frameshift (“W738fsX746” in Table 1). Thismutation is caused by, for example, a mutation of guanine (G) atposition 2213 in the nucleotide sequence of the sodium ion channel a 1subunit gene represented by SEQ ID NO. 2, preferably a deletion ofguanine (G) at position 2213 (G2213de1).

Yet another embodiment is a mutation of leucine (L) at position 990 ofthe amino acid sequence of sodium ion channel α1 subunit represented bySEQ ID NO. 1, preferably by a mutation of leucine (L) at position 990 tophenylalanine (F) (“L990F” in Table 1). This mutation is caused by, forexample, a mutation of guanine (G) at position 2970 of the nucleotidesequence of sodium ion channel α 1 subunit gene represented by SEQ IDNO. 2, preferably a substitution of guanine (G) at position 2970 withthymine (T) (G2970T).

Yet another embodiment is a mutation of glycine (G) at position 163 ofthe amino acid sequence of sodium ion channel α1 subunit represented bySEQ ID NO. 1, preferably a mutation of glycine (G) at position 163 toglutamic acid (E) (“G163E” in Table 1). This mutation is caused by, forexample, a mutation of guanine (G) at position 488 of the nucleotidesequence of sodium ion channel α 1 subunit gene represented by SEQ IDNO. 2, preferably a substitution of guanine (G) at position 488 withadenine (A) (G488A).

Yet another embodiment is a mutation of alanine (A) at position 1662 ofthe amino acid sequence of sodium ion channel α 1 subunit represented bySEQ ID NO. 1, preferably a mutation of alanine (A) at position 1662 tovaline (V) (“A1662V” in Table 1). This mutation is caused by, forexample, a mutation of cytosine (C) at position 4985 in the nucleotidesequence of sodium ion channel α 1 subunit gene represented by SEQ IDNO. 2, preferably by a substitution of cytosine (C) at position 4985with thymine (T) (C4985T).

Yet another embodiment is a mutation of lysine (K) at position 1057 ofthe amino acid sequence of sodium ion channel α1 subunit represented bySEQ ID NO. 1, preferably a mutation causing generation of a stop codonat position 1073 by frameshift (“K1057fsX1073” in Table 1). Thismutation is caused by, for example, a mutation of 14 nucleotides(AGAAAGACAGTTGT) between positions 3170 to 3183 of the nucleotidesequence of sodium ion channel α1 subunit gene represented by SEQ ID NO.2, preferably a substitution of the 14 nucleotides between the positions3170 to 3183 with TCATTCTGTATG.

It is needless to say that the mutation on the α-subunit type 1 of thevoltage-gated sodium ion channel Na_(V)1.1 is not limited to themutations exemplified above.

Examples of mutations on a calcium ion channel al subunit encompass,more specifically, a mutation on methionine (M) at position 249 of anamino acid sequence of calcium ion channel α1 subunit represented by SEQID NO. 3, preferably a mutation on methionine (M) at position 249 tolysine (K) (“M249K” in Table 2). This mutation is caused by, forexample, a mutation on thymidine (T) at position 746 of the nucleotidesequence of calcium ion channel α1 subunit gene represented by SEQ IDNO. 4, preferably a mutation on thymidine (T) at position 746substituted with adenine (A) (T746A).

Moreover, another embodiment is a mutation on glutamic acid (E) atposition 921 of the amino acid sequence of calcium ion channel α 1subunit represented by SEQ ID NO. 3, preferably a mutation on glutamicacid (E) at position 921 to aspartic acid (D) (“E921D” in Table 2). Thismutation is, for example, caused by a mutation on adenine (A) atposition 2762 of the nucleotide sequence of calcium ion channel α 1subunit gene represented by SEQ ID NO. 4, preferably a substitution ofadenine (A) at position 2762 with cytosine (C) (A2762C).

Yet another embodiment is a mutation on glutamic acid (E) at position996 of the amino acid sequence of calcium ion channel α 1 subunitrepresented by SEQ ID NO. 3, preferably a mutation on glutamic acid (E)at position 996 to valine (V) (“E996V” in Table 2). This mutation is,for example, caused by a mutation on adenine (A) at position 2987 of thenucleotide sequence of the calcium ion channel α 1 subunit generepresented by SEQ ID NO. 4, preferably a substitution of adenine (A) atposition 2987 with thymine (T) (A2987T).

Yet another embodiment is a mutation on arginine (R) at position 1126 ofthe amino acid sequence of calcium ion channel α 1 subunit representedby SEQ ID NO. 3, preferably a mutation on arginine (R) at position 1126to histidine (H) (“R1126H” in Table 2). This mutation is, for example,caused by a mutation on guanine (G) at position 3377 of the nucleotidesequence of calcium ion channel α 1 subunit gene represented by SEQ IDNO. 4, preferably a substitution of guanine (G) at position 3377 withadenine (A) (G3377A).

Yet another embodiment is a mutation on arginine (R) at position 2201 ofthe amino acid sequence of calcium ion channel α 1 subunit representedby SEQ ID NO. 3, preferably a mutation on arginine (R) at position 2201to glutamine (Q) (“R2201Q” in Table 2). This mutation is, for example,caused by mutation on guanine (G) at position 6602 of the nucleotidesequence of calcium ion channel α 1 subunit gene represented by SEQ IDNO. 4, preferably by a substitution of guanine (G) at position 6602 withadenine (A) (G6602A).

Yet another embodiment is a mutation on glycine (G) at position 1108 ofthe amino acid sequence of calcium ion channel α 1 subunit representedby SEQ ID NO. 3, preferably a mutation on glycine (G) at position 1108to serine (S) (“G1108S” in Table 2). This mutation is, for example,caused by a mutation on guanine (G) at position 3322 of the nucleotidesequence of calcium ion channel α 1 subunit gene represented by SEQ IDNO. 4, preferably a substitution of guanine (G) at position 3322 withadenine (A) (G3322A).

Yet another embodiment is a mutation on alanine (A) at position 924 ofthe amino acid sequence of calcium ion channel α 1 subunit representedby SEQ ID NO. 3, preferably a mutation of alanine (A) at position 924 toglycine (G) (“A924G” in Table 2). This mutation is, for example, causedby a mutation on cytosine (C) at position 2771 of the nucleotidesequence of calcium ion channel α 1 subunit gene represented by SEQ IDNO. 4, preferably a substitution of cytosine (C) at position 2771 withguanine (G) (C2771G).

Yet another embodiment is a mutation on glycine (G) at position 266 ofthe amino acid sequence of calcium ion channel α 1 subunit representedby SEQ ID NO. 3, preferably a mutation on glycine (G) at position 266 toserine (S) (“G2665” in Table 2). This mutation is, for example, causedby a mutation on guanine (G) at position 796 of the nucleotide sequenceof calcium ion channel α 1 subunit gene represented by SEQ ID NO. 4,preferably by a substitution of guanine (G) at position 796 with adenine(A) (G796A).

Yet another embodiment is a mutation on lysine (K) at position 472 ofthe amino acid sequence of calcium ion channel α 1 subunit representedby SEQ ID NO. 3, preferably a mutation on lysine (K) at position 472 toarginine (R) (“K472R” in Table 2). This mutation is, for example, causedby a mutation on adenine (A) at position 1415 of the nucleotide sequenceof calcium ion channel α 1 subunit gene represented by SEQ ID NO. 4,preferably by a substitution of adenine (A) at position 1415 withguanine (G) (A1415G).

Yet another embodiment is a deletion of an amino acid at positions 2202to 2205 of the amino acid sequence of calcium ion channel α 1 subunitrepresented by SEQ ID NO. 3 (“de12202-2205” in Table 2). This mutationis, for example, caused by a mutation on ACCAGGAGCGGG of positions 6605to 6616 of the nucleotide sequence of calcium ion channel a 1 subunitgene represented by SEQ ID NO. 4, preferably a deletion of ACCAGGAGCGGGat positions 6605 to 6616 (de16605-6616).

It is needless to say that the mutations related to the functionabnormality of voltage-gated calcium ion channel Ca_(V)2.1 is notlimited to the mutations exemplified above.

The mutations on the foregoing sodium ion channel a 1 subunit and themutations on the foregoing calcium ion channel α 1 subunit are organizedinto Table 1 and Table 2.

TABLE 1 Mutations on sodium ion channel α1 subunit 1289de1F, G177R,Q1450R, T1539P, A1002fsX1009, G1880fsX1881, R1648C, T1909I,A1429fsX1443, intron 21, R377L, V1390M, A1662V, K1027X, R501fsX543,V212A, A1685D, K1057fsX1073, R712X, W1271X, E788K, K547fsX570, R865X,W1434R, Exon10*, L1265P, R931C, W738fsX746, F902C, L990F, S1574X,N1417H, G163E, P707fsX714, S607fsX622, G1674R, Q1277X, T1082fsX1086,Exon10* exon deletion detected by MLPA

TABLE 2 Mutations on calcium ion channel α1 subunit A924G, E996V, K472R,del 2202-2205, G1108S, R1126H, E921D, G266S, R2201Q, M249K

In the assessment method according to the present invention, it ispreferable that the mutation on sodium ion channel α 1 subunit is, morespecifically, at least one mutation shown in Table 1, and the mutationon calcium ion channel al subunit is, more specifically, at least onemutation shown in Table 2.

The assessment method according to the present invention is not limitedin particular of how the presence of a mutation is detected for both thesodium ion channel a 1 subunit and the calcium ion channel α 1 subunit,and any method conventionally known may be used.

Examples of methods for detecting the presence of the mutation for boththe sodium ion channel α 1 subunit gene and the calcium ion channel α 1subunit gene encompass mutation detecting methods such as DNA sequencingmethod using PCR, SSCP method (Single strand conformation polymorphism),DHPLC method (denaturing high performance liquid chromatography);polymorphism detecting methods using real-time PCR or DNA chip; methodof detecting micro-deletion of exons of a gene; and Northern blotting,RT-PCR, Real-time PCR, and cDNA array, each of which detect an increaseand decrease of mRNA. Moreover, when the presence of mutation is to bedetected for both of sodium ion channel α 1 subunit protein and calciumion channel α 1 subunit protein, a method such as Western blotting,immunostaining, protein array or the like may be used.

The following provides more specific descriptions, by separating intothe following embodiments: (A) an embodiment detecting a gene mutationwith use of a genomic DNA included in a sample taken from a subject, (B)an embodiment detecting a gene mutation with use of mRNA (cDNA) includedin a sample taken from a subject, and (C) an embodiment detecting aprotein mutation with use of a protein included in a sample taken from asubject.

(A) Embodiment Using Genomic DNA

In the embodiment detecting a gene mutation with use of a genomic DNAincluded in a sample taken from a subject, first, a genomic DNA isextracted from the sample taken from the subject, by a conventionallyknown method.

The “sample taken from the subject” is not limited in particular, andany sample from which a genomic DNA is extractable can be used. Morespecifically, a sample of blood, oral mucosa cells, bone marrow fluid,hair, various organs, peripheral lymphocytes, synovial cells or the likecan be used. Moreover, cells taken from the subject may be cultured anda genomic DNA may be extracted from its proliferated cells.

Moreover, the extracted genomic DNA may be used upon amplification by agene amplification method generally performed, for example, PCR(Polymerase Chain Reaction), NASBA (Nucleic acid sequence basedamplification), TMA (Transcription-mediated amplification), SDA (StrandDisplacement Amplification), LAMP (Loop-Mediated IsothermalAmplification), and ICAN (Isothermal and Chimeric primer-initiatedAmplification of Nucleic acids).

The method of detecting the presence of mutation for both the sodium ionchannel α 1 subunit gene and the calcium ion channel α 1 subunit genewith use of a sample including a genomic DNA prepared as such is notlimited in particular, and examples encompass allele-specificoligonucleotide probe method, Oligonucleotide Ligation Assay, PCR-SSCP,PCR-CFLP, PCR-PHFA, invader method, RCA (Rolling Circle Amplification),Primer Oligo Base Extension, and like methods.

More specifically, a polynucleotide for detecting a mutation onα-subunit type 1 of the voltage-gated sodium ion channel Na_(V)1.1 and apolynucleotide for detecting a mutation on α-subunit type 1 of thevoltage-gated calcium ion channel Ca_(V)2.1 are used to detect, from thegenomic DNA, the presence of a mutation for both the sodium ion channelα 1 subunit gene and the calcium ion channel α1 subunit gene.

The “polynucleotide for detecting a mutation on α-subunit type 1 ofvoltage-gated sodium ion channel Na_(V)1.1” is indicative of apolynucleotide having a nucleotide sequence complementary to a setregion in a sodium ion channel al subunit gene (e.g. a region includingan exon, or boundary region between an exon and an intron). The“polynucleotide for detecting a mutation on α-subunit type 1 ofvoltage-gated calcium ion channel Ca_(V)2.1” is indicative of apolynucleotide having a nucleotide sequence complementary to a setregion in the calcium ion channel α1 subunit gene (e.g. a regionincluding an exon, or a boundary region between an exon and an intron).

The “polynucleotide for detecting a mutation on α-subunit type 1 ofvoltage-gated sodium ion channel Na_(V)1.1” is, more specifically, apolynucleotide having a nucleotide sequence represented by any one ofSEQ ID NOs.: 5, 6, and 9 to 62, for example. Moreover, the“polynucleotide for detecting a mutation on α-subunit type 1 ofvoltage-gated calcium ion channel Ca_(V)2.1” is, more specifically, apolynucleotide having a nucleotide sequence represented by any one ofSEQ ID NOs.: 7, 8, and 63 to 143.

Two kinds of the polynucleotides may be used in combination as a primerpair, or one kind may be used as a probe. When the two kinds are used incombination as a primer pair, the polynucleotides may be used incombinations as exemplified in Examples described later.

When two kinds of the polynucleotides are used in combination as aprimer pair, it is possible, for example, to amplify a set region in thegene by PCR with use of a corresponding primer pair, and thereafter,directly sequence the obtained PCR product, to detect the presence ofthe mutation in the gene.

Moreover, two kinds of fluorescence-labeled polynucleotides may be usedas a primer pair, to amplify a set region of the gene by PCR, performgel electrophoresis or capillary electrophoresis with the obtained PCRproduct, and study a strength of the signals, so as to detect thepresence of a mutation in the gene.

Moreover, when one kind of the polynucleotides is to be solely used as aprobe, the presence of the mutation on the gene can be detected by, forexample, digesting the genomic DNA with an appropriate restrictionenzyme and detecting a difference in size of the digested genomic DNAfragment by Southern blotting or the like.

As such, by detecting the presence of mutations for both the sodium ionchannel α 1 subunit gene and calcium ion channel α 1 subunit gene withuse of the genomic DNA included in the sample taken from the subject, itis possible to obtain data for assessing a potential for development ofDravet syndrome in the subject. More specifically, when a mutation isfound on both the sodium ion channel α 1 subunit gene and the calciumion channel α 1 subunit gene in the obtained data, it can be assessedthat the subject has a high potential for development of Dravetsyndrome.

The primer pair and probe used in the method of detecting the mutationmay be prepared by a DNA synthesizer or the like, as in law of the art.

(B) Embodiment Using mRNA (cDNA)

In the embodiment of detecting a mutation with use of mRNA included in asample taken from the subject, first, mRNA is extracted from a sampletaken from the subject, with use of a conventionally known method.

The “sample taken from the subject” is not limited in particular, andany sample can be used as long as mRNA can be extracted therefrom and agene that can be subjected to the detection of a mutation is expressedor is possibly expressed. The “sample taken from the subject” ispreferably, for example, a peripheral blood leukemic cell, dermalfibroblast, oral mucosa cell, neuron, or muscle cell, each of a patient.

Subsequently, cDNA is prepared from the extracted mRNA by reversetranscription reaction. Furthermore, if necessary, the obtained cDNA maybe amplified by a gene amplification method generally performed, forexample PCR (Polymerase Chain Reaction), NASBA (Nucleic acid sequencebased amplification), TMA (Transcription-mediated amplification), SDA(Strand Displacement Amplification), LAMP (Loop-Mediated IsothermalAmplification), and ICAN (Isothermal and Chimeric primer-initiatedAmplification of Nucleic acids).

The method of detecting the presence of the mutation for both the sodiumion channel α 1 subunit gene and calcium ion channel α 1 subunit genewith use of a sample including cDNA prepared as such is not limited inparticular; whether or not a gene mutation is present in a subject thatis subjected to mutation detection may be detected with use of a similarmethod as with a case in which a gene mutation is detected with use of agenomic DNA, as described in the foregoing “(A) Embodiment using genomicDNA”.

By detecting the presence of the mutation for both the sodium ionchannel α 1 subunit gene and calcium ion channel α1 subunit gene withuse of mRNA included in the sample that is taken from the subject, it ispossible to obtain data for assessing a potential for development ofDravet syndrome in the subject. More specifically, when a mutation isfound in both the sodium ion channel α 1 subunit gene and the calciumion channel α 1 subunit gene in the obtained data, it can be assessedthat the subject has a high potential for the development of Dravetsyndrome.

(C) Embodiment Using Protein

In the embodiment of detecting a mutation using protein included in thesample taken from a subject, first, protein is extracted from the sampletaken from the subject with use of a conventionally known method.

The sample taken from the subject is not limited in particular, and maybe any sample from which protein is extractable and in which both ofsodium ion channel a 1 subunit protein and calcium ion channel α 1subunit protein are expressed or is possibly expressed.

The method of detecting the presence of mutation for both the sodium ionchannel α 1 subunit protein and the calcium ion channel α 1 subunitprotein with use of the sample including the protein prepared asdescribed above is not limited in particular, and for example anantibody which specifically recognizes just a protein having a setmutation may be prepared, to detect the mutation by ELISA or Westernblotting using that antibody. In the present specification, the term“protein” may be used replaceable with “polypeptide” or “peptide”.

Moreover, mutation may be detected by isolating a protein to besubjected to the mutation detection from the sample including theforegoing protein, and digesting the isolated protein with an enzyme orthe like directly or if necessary, with use of a protein sequencer or amass spectrometer. Alternatively, the mutation may be detected on thebasis of an isoelectric point of the isolated protein.

As such, by detecting the presence of a mutation for both of the sodiumion channel α1 subunit protein and the calcium ion channel α1 subunitprotein with use of a protein included in the sample taken from thesubject, it is possible to obtain data for assessing potential fordevelopment of Dravet syndrome in the subject. More specifically, when amutation is found on both the sodium ion channel α1 subunit protein andthe calcium ion channel α 1 subunit protein in the obtained data, it ispossible to assess that the subject has a high potential for developmentof Dravet syndrome.

(1-2. Step of Detecting Change in Activity)

In the present specification, the “step of detecting change in activity”is indicative of a step of detecting whether activity of thevoltage-gated sodium ion channel Na_(V)1.1 has changed and a step ofdetecting whether activity of the voltage-gated calcium ion channelCa_(V)2.1 has changed.

As described in Examples later described, it is considered that thechange in activity in both the voltage-gated sodium ion channelNa_(V)1.1 and the voltage-gated calcium ion channel Ca_(V)2.1, caused bythe mutations on the sodium ion channel α1 subunit and on the calciumion channel α1 subunit, is related to the development of Dravetsyndrome. Hence, although the mutation on the sodium ion channel α1subunit is not particularly limited in its position, it is preferablethat the mutation is on a position that causes a change in the activityof the voltage-gated sodium ion channel Na_(V)1.1. Moreover, althoughthe mutation on the calcium ion channel α1 subunit is not particularlylimited in its position, it is preferable that the mutation is on aposition that causes a change in the activity of the voltage-gatedcalcium ion channel Ca_(V)2.1.

Here, the activity of the voltage-gated sodium ion channel Na_(V)1.1 is,more specifically, an activity to allow transmission of sodium ion (Na+)into the cell by depending on membrane potential. The change in activityof the voltage-gated sodium ion channel Na_(V)1.1 is not limited inparticular, and may be an increase of activity or may be a decrease inactivity. Namely, the change is sufficiently one that shows anabnormality in the activity of the voltage-gated sodium ion channelNa_(V)1.1.

In the present specification, “the activity of the voltage-gated sodiumion channel Na_(V)1.1 is changed” indicates that an activity of a mutantvoltage-gated sodium ion channel Na_(V)1.1 including the sodium ionchannel α1 subunit on which the mutation is present is of a value havinga statistically significant difference based on a significant test ascompared to an activity of a wild-type voltage-gated sodium ion channelNa_(V)1.1, and preferably indicates that p is equal to or smaller than0.05 by Student's t-test.

Moreover, the activity of the voltage-gated calcium ion channelCa_(V)2.1 is, more specifically, an activity that causes transmission ofcalcium ion (Ca²⁺) into the cell to be membrane voltage-gated. Thechange in function of the voltage-gated calcium ion channel Ca_(V)2.1 isnot particularly limited, and may be the increase of activity or thedecrease in activity. Namely, the change is sufficiently one that showsabnormality of the activity of the voltage-gated calcium ion channelCa_(V)2.1.

In the present specification, “the activity of the voltage-gated calciumion channel Ca_(V)2.1 is changed” indicates that the activity of amutant voltage-gated calcium ion channel Ca_(V)2.1 including the calciumion channel al subunit on which a mutation is present is of a valuehaving a statistically significant difference based on a significanttest as compared to an activity of a wild-type voltage-gated calcium ionchannel Ca_(V)2.1, and preferably indicates that p is equal to orsmaller than 0.05 by Student's t-test.

An example of a method of detecting that the activity of thevoltage-gated sodium ion channel Na_(V)1.1 is changed by the mutationis, for example, (i) coexpressing, in a culture cell with use of aexpression vector or the like, a sodium ion channel α1 subunit gene onwhich a mutation is present with a wild-type gene (β₁ subunit gene andβ₂ subunit gene) that encodes a subunit (β₁ subunit and β₂ subunit)other than the α1 subunit, which wild-type gene makes up thevoltage-gated sodium ion channel Na_(V)1.1, (ii) measuring an activityof the voltage-gated sodium ion channel Na_(V)1.1 on which a mutation ispresent with use of the obtained cultured cell, and (iii) comparing theactivity with an activity of the wild-type voltage-gated sodium ionchannel Na_(V)1.1, to confirm whether the activity of the voltage-gatedsodium ion channel Na_(V)1.1 is changed. The method of measuring theactivity of the voltage-gated sodium ion channel Na_(V)1.1 is notparticularly limited, however it is possible to use the conventionallyknown patch clamping, imaging with use of a fluorescence probe, or likemethod.

An example of a method of detecting that the activity of thevoltage-gated calcium ion channel Ca_(V)2.1 is changed by mutation is by(i) coexpressing, in a culture cell with use of an expression vector orthe like, a calcium ion channel al subunit gene on which a mutation ispresent with a wild-type gene (β subunit gene, γ subunit gene, and α2δsubunit gene) that encodes a subunit (β subunit, γ subunit, and α2δsubunit) other than the α1 subunit, which wild-type gene makes up thevoltage-gated calcium ion channel Ca_(V)2.1, (ii) measuring, with theobtained cultured cell, an activity of the voltage-gated calcium ionchannel Ca_(V)2.1 on which the mutation is present, and (iii) comparingthe activity with an activity of the wild-type voltage-gated calcium ionchannel Ca_(V)2.1, to confirm whether the activity of the voltage-gatedcalcium ion channel Ca_(V)2.1 is changed. The method of measuring theactivity of the voltage-gated calcium ion channel Ca_(V)2.1 is notlimited in particular, however it is possible to use the conventionallyknown patch clamping, imaging using an optical probe, a calciumindicator, or a caged compound, for example.

The assessment method according to the present invention, since itincludes the foregoing configuration, it is possible to obtain data forassessing a potential for development of Dravet syndrome in the subject.Hence, with the assessment method according to the present invention, itis possible to find out, with high accuracy and at an early stage,Dravet syndrome having the unfavorable prognosis, which thus allows forpreparing a treatment management system by an epilepsy specialist froman earlier stage for a Dravet syndrome patient. As a result, it ispossible to improve treatment intervention of the patient, reduce themental burden on their families, and reduce the economical burden.Furthermore, it is possible to provide appropriate treatment for thepatient of Dravet syndrome; this hence reduces medical fees.

2. Kit According to the Present Invention

The present invention also encompasses a kit for assessing the potentialfor development of Dravet syndrome, with use of the assessment methodaccording to the present invention (hereinafter, also referred simply as“kit according to the present invention”).

The kit according to the present invention is not limited in itsspecific configuration in particular as long as it includes at least areagent for detecting the presence of mutation on α-subunit type 1 ofthe voltage-gated sodium ion channel Na_(V)1.1 and a reagent fordetecting the presence of mutation on α-subunit type 1 of thevoltage-gated calcium ion channel Ca_(V)2.1.

As described in “1. Assessment method according to the presentinvention”, ways considered to detect the presence of mutation for bothof α-subunit type 1 of the voltage-gated sodium ion channel Na_(V)1.1and α-subunit type 1 of the voltage-gated calcium ion channel Ca_(V)2.1are (A) detecting a gene mutation with use of a genomic DNA included ina sample taken from a subject, or (B) detecting a gene mutation with useof mRNA (cDNA) included in a sample taken from the subject.

Hence, in order to detect a mutation using a genomic DNA included in thesample taken from the subject or mRNA (cDNA) included in the sampletaken from the subject, the kit according to the present inventionincludes a polynucleotide being used for determining a mutation onα-subunit type 1 of voltage-gated sodium ion channel Na_(V)1.1; and apolynucleotide being used for determining a mutation on α-subunit type 1of voltage-gated calcium ion channel Ca_(V)2.1. Such polynucleotides canbe used as, for example, a primer pair or a probe. These polynucleotidesmay be included solely or may be included as a combination of aplurality thereof.

The kit according to the present invention encompasses (A) a kit fordetecting a mutation with use of a genomic DNA included in a sampletaken from a subject and (B) a kit for detecting a mutation with use ofa mRNA (cDNA) included in a sample taken from a subject. The followingspecifically describes the reagents included in the embodiments of thekits in (A) or (B).

(A) Kit for detecting mutation with use of genomic DNA included insample taken from subject

For example, a configuration of the sodium ion channel α1 subunit andthe calcium ion channel α 1 subunit may include a primer pair designedso as to allow amplification of the genomic DNA of each of the genes ora part of its region, or may include a probe designed so that one ofgenomic DNA of its mutant type or wild-type can be specificallydetected. These polynucleotides are as described in the foregoing (A)Embodiment using genomic DNA in “1. Assessment method according to thepresent invention”, so hence its description has been omitted here.

Furthermore, such a kit may be configured to include, in addition to theprimer pair or probe, a combination of one or more reagent necessary fordetecting the presence of the mutation on the gene, such as a reagentused in PCR, Southern blotting, and nucleic acid sequencing.

The reagent is selected and employed as appropriate in accordance withthe detection method of the present invention, and examples thereof aredATP, dCTP, dTTP, dGTP, DNA polymerase and the like. Furthermore, thekit according to the present invention may include a suitable buffersolution and a washing solution that can be used in the PCR, Southernblotting, and nucleic acid sequencing.

(B) Kit detecting mutation with use of mRNA (cDNA) included in sampletaken from subject For example, a configuration of the sodium ionchannel α1 subunit and the calcium ion channel α 1 subunit may include aprimer pair designed so as to allow amplification of the cDNA of each ofthe genes or a part of its region, or include a probe designed so thatone of mRNA of its mutant type or wild-type can be specificallydetected. These polynucleotides are as described in (B) Embodiment usingmRNA (cDNA) in “1. Assessment method according to the presentinvention”, so hence its description has been omitted here.

Furthermore, such a kit may be configured to include, in addition to theprimer pair or probe, a combination of one or more reagent necessary fordetecting the presence of a mutation on the gene, such as a reagent usedin RT-PCR, Northern blotting, nucleic acid sequencing or the like.

The reagent is selected and employed as appropriate in accordance withthe detection method of the present invention, and examples thereof aredATP, dCTP, dTTP, dGTP, DNA polymerase and the like. Furthermore, thekit according to the present invention may include a suitable buffersolution and a washing solution that can be used in RT-PCR, Northernblotting, and nucleic acid sequencing.

The kit according to the present invention may include the exemplifiedconfiguration in any combination. Furthermore, the kit may include otherreagents other than the reagents exemplified above.

As described in the item “1. Assessment method according to the presentinvention”, in order to detect the presence of mutation for both thesodium ion channel a 1 subunit and the calcium ion channel α 1 subunit,it is further considerable to (C) detect the mutation with use of aprotein included in the sample taken from a subject.

Therefore, the kit according to the present invention may include, forexample, an antibody that specifically bonds to just the wild-type ormutant protein among the proteins of the sodium ion channel α 1 subunitand the calcium ion channel α 1 subunit. Furthermore, the configurationmay be one which, in addition to the antibody, includes one or morereagent in combination, which reagent is used for ELISA or Westernblotting.

Furthermore, the kit according to the present invention may include areagent used for measuring activity of the voltage-gated sodium ionchannel Na_(V)1.1, a reagent used for measuring activity of thevoltage-gated calcium ion channel Ca_(V)2.1, or the like.

With use of the kit according to the present invention as describedabove, it is possible to easily obtain data for assessing the potentialfor development of Dravet syndrome in the subject. A subject to whichthe kit may be applied is not particularly limited, however ispreferably applied to infants or children.

3. Model Animal of Dravet Syndrome According to the Present Inventionand its Production Method

The present invention encompasses a model animal of Dravet syndrome, andits production method.

(3-1. Model Animal of Dravet Syndrome According to the PresentInvention)

The model animal of Dravet syndrome according to the present inventionhas a mutation on both the sodium ion channel α 1 subunit and thecalcium ion channel α 1 subunit. The mutation on the sodium ion channelα 1 subunit and the mutation on the calcium ion channel α 1 subunit areas described in the item “1. Assessment method according to the presentinvention” described above, so therefore specific descriptions thereofare omitted here.

It is preferable in the model animal of the Dravet syndrome that boththe activity of the voltage-gated sodium ion channel Na_(V)1.1 and theactivity of the voltage-gated calcium ion channel Ca_(V)2.1 are changedas compared to a wild-type animal. This change in activity is notparticularly limited, and may be an increase of activity or may be adecrease in activity. The method of confirming whether or not anactivity of the voltage-gated sodium ion channel Na_(V)1.1 of the modelanimal of Dravet syndrome according to the present invention is changedfrom that of a wild-type, and the method of confirming whether or not anactivity of the voltage-gated calcium ion channel Ca_(V)2.1 of the modelanimal of Dravet syndrome according to the present invention is changedfrom that of a wild-type, are both not particularly limited. Forexample, with an individual of a model animal of Dravet syndromeaccording to the present invention or cells collected from the modelanimal of Dravet syndrome according to the present invention,confirmation may be made by measuring the activity by use of theconventionally known patch clamping, slice patching, imaging with use offluorescence probe and like method.

The model animal of Dravet syndrome according to the present inventionhas the mutation on both the sodium ion channel α 1 subunit and thecalcium ion channel α 1 subunit, so therefore develops Dravet syndrome.Such a model animal of Dravet syndrome can be used advantageously forclarification of the development mechanism of the intractable Dravetsyndrome, and for development of medicament for Dravet syndrome.

In the present specification, “model animal” denotes an experimentanimal used for developing a prevention method or treatment againsthuman diseases, and more specifically is a non-human mammal such as amouse, rat, rabbit, monkey, goat, pig, sheep, cow, or dog, and othervertebrates.

(3-2. Production Method of Model Animal of Dravet Syndrome According tothe Present Invention)

A method of producing a model animal of Dravet syndrome, according tothe present invention, includes: introducing a mutation on sodium ionchannel α 1 subunit and introducing a mutation on calcium ion channel α1 subunit.

More specifically, a mutation can be introduced on each of the genes bymanipulating the gene of the model animal. Here, the “manipulating thegene of the model animal” intends to mean manipulation of a gene of amodel animal by use of a conventionally known gene manipulationtechnique. More specifically, this encompasses all of destruction of agene of the model animal, an introduction of a mutation to that gene, asubstitution of that gene with a mutant gene, and furthermore,introduction of a foreign gene into the model animal, and crossing ofmodel animals.

The production method according to the present invention of the modelanimal of Dravet syndrome may include steps other than those describedabove. Specific steps, materials, conditions, used devices, usedequipment and the like are not limited in particular.

With the production method according to the present invention of a modelanimal of Dravet syndrome, it is possible to produce a model animaldeveloped in Dravet syndrome by manipulating genes of a model animal sothat a mutation is introduced into the genes of the sodium ion channel a1 subunit and the calcium ion channel α 1 subunit.

4. Cells According to the Present Invention and its Production Method

The present invention also encompasses cells having a mutation on boththe sodium ion channel α 1 subunit and the calcium ion channel α 1subunit, and its production method.

(4-1. Cell According to the Present Invention)

The cell according to the present invention is a cell having a mutationon both the sodium ion channel α 1 subunit and the calcium ion channel α1 subunit. The mutation on the sodium ion channel α 1 subunit and themutation on the calcium ion channel α 1 subunit are as described in theitem “1. Assessment method according to the present invention” describedabove, so therefore specific description thereof have been omitted here.

The cell according to the present invention intends to mean experimentalculture cells having a mutation on both the sodium ion channel α 1subunit and the calcium ion channel α1 subunit. More specifically, thecell is an experimental culture cell derived from a mammal such as ahuman, mouse, rat, hamster, rabbit, monkey and the like, and othervertebrates.

It is preferable that with such a cell, both of activity of thevoltage-gated sodium ion channel Na_(V)1.1 and activity of thevoltage-gated calcium ion channel Ca_(V)2.1 are changed. This change inactivity is not particularly limited, and may be an increase of activityor a decrease in activity. The method of confirming whether or not theactivity of the voltage-gated sodium ion channel Na_(V)1.1 of the cellaccording to the present invention is changed from that of a wild-type,and a method of confirming whether or not the activity of both of thevoltage-gated calcium ion channel Ca_(V)2.1 of the cell according to thepresent invention is changed from that of the wild-type are as describedin “1. Assessment method according to the present invention” describedabove, so hence specific description thereof have been omitted here.

Such a cell can be used for clarification of a development mechanism ofthe intractable Dravet syndrome, and for the development in medicamentfor Dravet syndrome. For example, it is possible to suitably use thisfor screening of a drug for treating Dravet syndrome. Namely, this cellcan also be said as a screening cell for a drug for treating Dravetsyndrome. Accordingly, the present invention also encompasses ascreening cell of a drug for treating Dravet syndrome (hereinafter,simply called “screening cell”), and its production method.

(4-2. Production Method of Cell According to Present Invention)

A method of producing a cell according to the present invention is amethod of producing a cell that has the foregoing properties, andincludes: introducing a mutation on a sodium ion channel α 1 subunit;and introducing a mutation on a calcium ion channel α 1 subunit. Morespecifically, the following three embodiments can be raised. Thefollowing three embodiments are described specifically below, howeverthe present invention is not limited to these.

(1) Method of Using Expression Vector Etc.

This method produces a cell that expresses a mutant voltage-gated sodiumion channel Na_(V)1.1 and mutant voltage-gated calcium ion channelCa_(V)2.1, with use of an expression vector or the like. Morespecifically described, in order to make a cell express the mutantvoltage-gated sodium ion channel Na_(V)1.1, for example, a sodium ionchannel a 1 subunit gene having a mutation that causes a change in anamino acid is coexpressed, in a culture cell that serves as a host, witha wild-type gene (β₁ subunit gene and β₂ subunit gene) making up thevoltage-gated sodium ion channel Na_(V)1.1, which wild-type gene encodesa subunit other than the α1 subunit (β₁ subunit and β₂ subunit), withuse of an expression vector or the like. This enables the cell toexpress the mutant voltage-gated sodium ion channel Na_(V)1.1 thatincludes the mutant sodium ion channel α 1 subunit.

Similarly, in order to make the cell express the mutant voltage-gatedcalcium ion channel Ca_(V)2.1, for example, a calcium ion channel α 1subunit gene having a mutation that causes a change in an amino acid iscoexpressed, in a culture cell that serves as a host, with a wild-typegene (β subunit gene, γ subunit gene, and α2δ subunit gene) making up avoltage-gated calcium ion channel Ca_(V)2.1, which wild-type geneencodes a subunit other than the α 1 subunit (β subunit, γ subunit, andα2δ subunit), with the expression vector or the like. This hence enablesthe cell to express a mutant voltage-gated calcium ion channel Ca_(V)2.1that includes the mutant calcium ion channel α 1 subunit.

At this time, it is preferable that the culture cell serving as a hostis a cell from which no voltage-gated sodium ion channel Na_(V)1.1 andthe voltage-gated calcium ion channel Ca_(V)2.1 is expressed. With useof such a cell, no effect is caused by the residing voltage-gated sodiumion channel Na_(V)1.1 and residing voltage-gated calcium ion channelCa_(V)2.1.

(2) Method of Using Artificial Mutation Introduction

This method introduces mutation for both of the sodium ion channel α 1subunit and the calcium ion channel a 1 in a culture cell expressingboth the voltage-gated sodium ion channel Na_(V)1.1 and thevoltage-gated calcium ion channel Ca_(V)2.1.

The method of introducing the mutation on the culture cell is notparticularly limited, and a conventionally known gene manipulationtechnique is used in combination as appropriate.

(3) Method of Using Model Animal of Dravet Syndrome According to thePresent Invention

This method extracts a tissue from the model animal of Dravet syndromeaccording to the present invention as described above, and prepares aculture cell from that tissue. The model animal of Dravet syndromeaccording to the present invention is as described in “3. Model animalof Dravet syndrome according to the present invention and its productionmethod”, and so therefore specific description thereof has been omittedhere. Of course, the “tissue” that is extracted is intended to mean atissue in which both the sodium ion channel α 1 subunit on which amutation is introduced and the calcium ion channel α 1 subunit on whicha mutation is introduced are expressed.

This hence allows for easy production of a cell that has a mutation onboth the sodium ion channel α 1 subunit and the calcium ion channel α 1subunit. The kinds of tissues extracted from the model animal of Dravetsyndrome is not limited in particular, and may be selected asappropriate depending on its purpose.

The method according to the present invention of producing a cell mayinclude steps other than the steps described above. Specific steps,materials, conditions, used devices, used equipment and the like are notlimited in particular.

5. Screening Method of Drug for Treating Dravet Syndrome

The model animal of Dravet syndrome according to the present inventionand the cell according to the present invention can be used indevelopment of a new treatment method and drug for treating Dravetsyndrome. Hence, the present invention encompasses a screening method ofa drug for treating Dravet syndrome, which screens a drug for treatingDravet syndrome (hereinafter, also called “screening method according tothe present invention”).

In the specification, an embodiment using a model animal of Dravetsyndrome according to the present invention and an embodiment using ascreening cell have been explained as embodiments of the screeningmethod according to the present application. However, the presentinvention is not limited to these embodiments.

Namely, for example, the embodiment may use another model animal ofDravet syndrome instead of the model animal of Dravet syndrome accordingto the present invention.

(1) Case of using model animal of Dravet syndrome according to thepresent invention

The method is sufficient as long as it includes administering acandidate agent to the model animal of Dravet syndrome according to thepresent invention, and assessing whether or not Dravet syndrome showsimprovement or is cured in the model animal of Dravet syndrome to whichthe candidate agent is administered.

Namely, according to the screening method of the drug for treatingDravet syndrome according to the present invention, a candidate agent isadministered to the model animal of Dravet syndrome, to assess whetheror not that candidate agent can serve as a drug for treating Dravetsyndrome in the model animal of Dravet syndrome to which the candidateagent is administered, by having the improvement or curing of Dravetsyndrome serve as an indicator.

The method of assessing whether or not Dravet syndrome is improved orcured in the model animal of Dravet syndrome to which the candidateagent is administered is not limited in particular, and is sufficientlyassessed by use of characteristic symptoms of Dravet syndrome asindicators. For example, it is possible to determine whether Dravetsyndrome is improved or cured by comparing a control animal not having amutation that causes an amino acid change on the sodium ion channel α1subunit gene and the calcium ion channel α 1 subunit gene (i.e. ananimal not having a mutation on both of α-subunit type 1 ofvoltage-gated sodium ion channel Na_(V)1.1 and α-subunit type 1 ofvoltage-gated calcium ion channel Ca_(V)2.1) with the model animal ofDravet syndrome according to the present invention, in terms of “bodytemperature at convulsion onset (convulsion threshold)”, “severityscore”, “duration of convulsion”, and the like each shown in theExamples later described.

The candidate agent is not limited in particular, however it ispreferable that it is a compound expectable of giving effect on theexpression of voltage-gated sodium ion channel Na_(V)1.1 and/orexpression of voltage-gated calcium ion channel Ca_(V)2.1, or a compoundexpectable of giving effect on the activity of the voltage-gated sodiumion channel Na_(V)1.1 and/or the activity of voltage-gated calcium ionchannel Ca_(V)2.1 (e.g. an inhibitor or candidate substance of aninhibitor, or an agonist or a candidate substance of an agonist, each ofwhich has effect on both the voltage-gated sodium ion channel Na_(V)1.1and the voltage-gated calcium ion channel Ca_(V)2.1).

Moreover, the candidate agent may be an expression plasmid vector or avirus vector that includes a polynucleotide made of a sodium ion channelα 1 subunit gene or a part of its nucleotide sequence. Moreover, thecandidate agent may be an expression plasmid vector or a virus vectorthat includes a polynucleotide made of the calcium ion channel α 1subunit gene or a part of its nucleotide sequence.

The method of administering such a candidate agent to the Dravetsyndrome model animal according to the present invention is not limitedin particular, and a suitable method is sufficiently selected fromconventionally known methods in accordance with physical properties ofthat candidate agent.

(2) Case of Using Screening Cell According to the Present Invention

The method at least includes administering a candidate agent to ascreening cell according to the present invention, and assessing whetheror not activity of voltage-gated sodium ion channel Na_(V)1.1 and/oractivity of voltage-gated calcium ion channel Ca_(V)2.1 in the screeningcell of a drug for treating Dravet syndrome to which the candidate agentwas administered, is changed.

Namely, with the screening method according to the present embodiment,it is possible to assess whether a candidate agent can serve as a drugfor treating Dravet syndrome, by administering the candidate agent tothe screening cell according to the present invention, based on anindicator of whether the activity of the voltage-gated sodium ionchannel Na_(V)1.1 and/or the activity of the voltage-gated calcium ionchannel Ca_(V)2.1 in the screening cell to which the candidate agent isadministered, is changed.

Moreover, the method of assessing, in the screening cell to which thecandidate agent is administered, whether or not the activity of thevoltage-gated sodium ion channel Na_(V)1.1 is changed and whether or notthe activity of the voltage-gated calcium ion channel Ca_(V)2.1 ischanged are not limited in particular, and the assessments aresufficiently carried out by use of an electrophysiologic measurementdevice, fluorescence observation device, or the like.

The candidate agent is not limited in particular, and similar substancesas those described in the foregoing “(1) Case of using model animal ofDravet syndrome according to the present invention” may be used.

The method of administering such a candidate agent to a cell accordingto the present invention is not limited in particular, and a suitablemethod based on the physical properties and the like of that candidateagent is selected and used from conventionally known methods.

It is preferable in the assessment method according to the presentinvention that the mutation on α-subunit type 1 of the voltage-gatedsodium ion channel Na_(V)1.1 is at least one of a mutation shown inTable 1, and

the mutation on α-subunit type 1 of the voltage-gated calcium ionchannel Ca_(V)2.1 is at least one of a mutation shown in Table 2.

It is preferable in the assessment method according to the presentinvention to further include:

detecting a change in activity of the voltage-gated sodium ion channelNa_(V)1.1; and

detecting a change in activity of the voltage-gated calcium ion channelCa_(V)2.1.

The present invention is not limited to the description of theembodiments above, but may be altered by a skilled person within thescope of the claims. An embodiment based on a proper combination oftechnical means disclosed in different embodiments is encompassed in thetechnical scope of the present invention.

EXAMPLES

The following describes more specifically of the present invention withuse of Examples, however the present invention is not limited to theExamples.

Example 1 Identification of Risk Factors for Predicting Development ofDravet Syndrome

DNA were extracted from peripheral blood of 47 Dravet syndrome patientswho visited Okayama University Hospital and/or its related hospitals,and mutations on various genes were analyzed. This study was performedupon receiving approval from Okayama University, Institutional ReviewBoard of Human Genome and Gene Analysis Research.

More specifically, a genomic DNA was extracted from peripheral blood ofa patient with use of a DNA extraction kit (WB kit; Nippon gene, Tokyo,Japan), and all exons were amplified by PCR. In PCR, a reaction solutionof 25 μl was used, which includes 50 ng of human genomic DNA, 20 μmol ofvarious primers, 0.8 mM of dNTPs, 1 reaction buffer, 1.5 mM of MgCl₂,and 0.7 units of AmpliTaq Gold DNA polymerase (Applied Biosystems,Foster City, Calif., USA). As to the nucleotide sequence (SEQ ID NOs.:9-62) of the primer pair used, see “Sequence of primers” describedlater.

An obtained PCR product was purified with use of PCR productspre-sequencing kit (Amersham Biosciences, Little Chalfont,Buckinghamshire, England). Subsequently, with use of Big Dye TerminatorFS ready-reaction kit (Applied Biosystems), a sequence reaction wasperformed, and with use of a fluorescence sequencer (ABI PRISM3100sequencer; Applied Biosystems), a nucleotide sequence of the obtainedPCR product was determined.

First, mutation analysis was performed of SCN1A gene that encodesα-subunit type 1 (also called “α1 subunit”) making up the voltage-gatedsodium ion channel Na_(V)1.1, for the 47 Dravet syndrome patients. As aresult, a mutation in the SCN1A gene was found in 38 patients out of the47 Dravet syndrome patients. For the 9 patients in which no mutation wasdetected, a further analysis was performed on the number of gene copiesof the SCN1A gene, with use of Multiplex Ligation-dependent ProbeAmplification (MLPA; MRC-Holland; SALSA MLPA kit P137). As a result, adeletion of exon 10 was detected in 1 patient. The number of patients inwhich no mutation of the SCN1A gene was found was 8. The mutationdetected in the SCN1A gene is as shown in Table 1.

Next, with use of the DNA of the 47 patients, gene analysis wasperformed for GABRG2 gene, CACNA1A gene, CACNB4 gene, SCN1B gene, andSCN3A gene. These genes encode proteins as follows:

GABRG2: GABAA receptor γ2 subunit gene

CACNA1A: α1 subunit of voltage-gated calcium ion channel Ca_(V)2.1

CACNB4: β4 subunit of voltage-gated calcium ion channel

SCN1B: β1 subunit of voltage-gated sodium ion channel

SCN3A: α3 subunit of voltage-gated sodium ion channel Na_(V)1.3

The nucleotide sequence (SEQ ID NOs.: 63-143) of the primer pair usedfor the gene analysis of the CACNA1A gene is shown in “Sequence ofprimers” described later.

As a result, various kinds of gene mutations were found in the CACNA1Agene that encodes α-subunit type 1 (also called “α1 subunit”) making upthe voltage-gated calcium ion channel Ca_(V)2.1 (see Table 2 and FIG.12).

Table 3 shows the gene mutations of SCN1A and CACNA1A that were detectedin the Dravet syndrome patients.

TABLE 3 SCN1A and CACNA1A gene mutations detected in Dravet syndromepatients P. No. SCN1A gene CACNA1A gene 1 G177R G266S 2 W738fsX746 K472R3 V1390M A924G 4 V212A E921D E996V 5 R377L E921D E996V 6 Deletion ofexon 10 E921D E996V (Exon10*) 7 P707fsX714 E921D E996V 8 R865X E921DE996V 9 F902C E921D E996V 10 T1082fsX1086 E921D E996V 11 Q1277X E921DE996V 12 Q1450R E921D E996V 13 A1685D E921D E996V 14 T1909I E921D E996VR1126H R2201Q 15 G163E R1126H R2201Q 16 K547fsX570 R1126H R2201Q 17S1574X R1126H R2201Q 18 R712X G1108S 19 R1648C G1108S 20 negative G1108S21 negative Del2202-2205 22 R501fsX543 negative 23 S607fsX622 negative24 E788K negative 25 R931C negative 26 R931C negative 27 L990F negative28 A1002fsX1009 negative 29 K1027X negative 30 K1057fsX1073 negative 31L1265P negative 32 W1271X negative 33 1289delF negative 34 Intron 21splicing negative error 35 A1429fsX1443 negative 36 W1434R negative 37T1539R negative 38 S1574X negative 39 G1674R negative 40 A1662V negative41 G1880fsX1881 negative 42 negative negative 43 negative negative 44negative negative 45 negative negative 46 negative negative 47 negativenegative P. No. Patient Number Exon10* exon deletion detected by MPLA

The following mutations are mutations of the CACNA1A gene detected thistime. These mutations were mutations that cause an amino acidsubstitution, mutations that cause no amino acid substitution, andintron mutations.

(1) Missense Mutations

G266S  1 case K472R  1 case E921D 11 cases A924G  1 case E996V 11 casesG1108S  3 cases R1126H  4 cases R2201Q  4 cases

(2) Deletion of Amino Acids

4 amino acid deletions (deletion 2202-2205) 1 case

(3) Gene Mutation Causing No Amino Acid Change in Exon E292E (rs16006),E394E (rs2248069), 15251 (rs16010), T698T (rs16016), R1023R (rs16025),F1291F (rs16030), T1458T (new SNP or mutation), S1472S (new SNP ormutation), V1890V (rs17846921), H2225H (rs16051)

(4) Gene Mutation in Intron

exon 1 upstream (rs16000), intron 1 (rs16003), intron 3 (rs17846942),intron 8 (rs2306348), intron 11 (rs10407951), intron 17 (rs16018),intron 39 (rs3816027), intron 40 (rs17846925), intron 42 (new SNP ormutation).

The missense mutations and deletion mutations detected in coding regionsof the CACNA1A gene shown in the foregoing (1), and (2) are shown inTable 4.

TABLE 4 Summary of mutations detected in coding region of CACNA1A geneCoding Region Mutation SNP Reg. Exon No. Amino acid type No. 1 Exon 6G266S Missense — 2 Exon 11 K472R Missense — 3 Exon 19 E921D Missensers16022 4 Exon 19 A924G Missense — 5 Exon 19 E996V Missense rs16023 6Exon 20 G1108S Missense rs16027 7 Exon 20 R1126H Missense — 8 Exon 47R2201Q Missense — 9 Exon 47 Del 2202-2205 Deletion — SNP Reg. No.:Single Nucleotide Polymorphism Registration Number

These mutations were compared and studied with a gene polymorphism(Single Nucleotide Polymorphism; SNP) database of NCBI (National Centerfor Biotechnology Information). As a result, it was found that 3 kindsof the mutations out of the 9 kinds of mutations were registered in theSNP database as gene polymorphism (Single Nucleotide Polymorphism; SNP).

The gene mutation shown in (3) and (4) were either a gene polymorphismregistered in the SNP database, or a new gene polymorphism or mutation.The registered number in the SNP database is shown in the brackets.

Out of the SNP already reported, the mutations which caused a change inthe amino acid were considered probably that although no seizure occursjust by that individual case having the CACNA1A gene SNP, but when anabnormality of SCN1A gene is simultaneously present, this is somewhatinvolved in the worsening of the symptom.

A comparison of patients having a mutation in either of the SCN1A geneand the CACNA1A gene or both of the SCN1A gene and CACNA1A gene, out ofthe 47 Dravet syndrome patients, resulted as follows.

Patients having a mutation on both SCN1A and CACNA1A: 19 cases

Patients having a mutation on just SCN1A: 20 cases

Patients having a mutation on just CACNA1A: 2 cases

Patients having no mutation on either of SCN1A or CACNA1A: 6 cases.

No reports whatsoever have been made regarding abnormalities in theCACNA1A gene of the patients of Dravet syndrome, until now. The resultof the present study shows that Dravet syndrome patients highlyfrequently has a mutation in SCN1A, i.e. a α1 subunit gene of thevoltage-gated sodium ion channel Na_(V)1.1, and in CACNA1A, i.e. a α1subunit gene of the voltage-gated calcium ion channel Ca_(V)2.1.

A literature disclosing that a mutation on a β4 subunit of thevoltage-gated calcium ion channel Ca_(V)2.1 (hereinafter, simplyreferred to as “calcium ion channel β4 subunit”) is involved with Dravetsyndrome (Iori Ohmori et al., Neurobiology of Disease 32 (2008) 349-354)describes that out of 38 patients in which a mutation was detected inthe sodium ion channel α 1 subunit, 1 Dravet syndrome patient had amutation on both the sodium ion channel α 1 subunit and the calcium ionchannel β4 subunit.

In comparison, out of 39 patients in which a mutation was detected onthe sodium ion channel α 1 subunit, the patients of Dravet syndromehaving a mutation on both the sodium ion channel α 1 subunit and thecalcium ion channel α1 subunit were 19 patients (6 patients whenexcluding patients having registered SNP that cause a change in an aminoacid in an exon). This result shows that by detecting the mutation forboth the sodium ion channel α 1 subunit and the calcium ion channel α 1subunit, the detection sensitivity of Dravet syndrome patientsdramatically increase as compared to detecting the mutation for both thesodium ion channel a 1 subunit and the calcium ion channel β4 subunit.

In the present specification, a nucleotide number in mRNA of the SCN1Agene and an amino acid number in a protein of SCN1A were made to be inline with GenBank accession No. AB093548; methionine, encoded by theinitiation codon (ATG), was numbered as the first amino acid, and theinitial A of the initiation codon was numbered as the first nucleotide.

Moreover, a genome sequence of the CACNA1A gene was in line with theGenBank accession number NC_(—)000019. The number of the nucleotide inmRNA of CACNA1A gene and the number of the amino acid in CACNA1A proteinwas made to be in line with the GenBank accession number NM 023035;methionine, encoded by the initiation codon (ATG), was numbered as theprimacy amino acid, and the initial A of the initiation codon wasnumbered as the primacy nucleotide.

Example 2 Study of Gene Mutation in Benign Febrile Seizure Patient

A study was performed of a SCN1A gene and CACNA1A gene abnormality in abenign febrile seizure patient. DNA was extracted from peripheral bloodof 50 patients of benign generalized epilepsy with febrile seizure plus(GEFS+), who visited Okayama University Hospital and/or its relatedhospitals, and mutations on various genes were analyzed. The DNAextraction, PCR amplification of the gene, and sequencing reactions wereperformed by the methods described above.

First, mutation analysis of voltage-gated sodium ion channel SCN1A genewas performed, which resulted in detecting gene mutation that causedamino acid changes in 6 patients. Next, mutation analysis was performedfor 9 kinds of mutations of missense mutations and deletion mutationsthat were detected in the coding region of the CACNA1A gene, whichresulted in detecting a mutation in 16 patients. Each of the mutationsare shown in Table 5.

TABLE 5 SCN1A and CACNA1A gene mutations detected in benign febrileseizure Patient No. SCN1A CACNA1A 1 2 M1856T 3 del 2202-2205 4 5 del2202-2205 6 R1575C 7 E921D E996V 8 E921D E996V 9 E921D E996V 10 11 12I1616T 13 14 15 16 17 18 E921D E996V 19 20 21 22 E921D E996V 23 E921DE996V 24 25 26 E921D E996V 27 28 E921D E996V 29 A924G 30 E921D E996V 3132 33 E921D E996V 34 G1108S 35 36 I1616T 37 I1616T 38 39 Y1769H 40 E921DE996V 41 42 43 44 45 46 47 48 E921D E996V 49 50

Out of the 50 benign epilepsy patients, it was confirmed that no patienthad mutations simultaneously on both SCN1A gene and CACNA1A gene.

The following shows a result of gene mutation analysis of a total of 97patients, of 47 malignant Dravet syndrome cases and 50 benign febrileseizure patient cases.

(1) As a result of screening patients having a mutation on the SCN1Agene among the 97 patients, 39 Dravet syndrome patients (39 cases out of47 cases) and 6 benign epilepsy patients (6 cases out of 50 cases) weredetected.

(2) As a result of screening patients having a mutation on both theSCN1A gene and CACNA1A gene out of the 97 patients, 19 Dravet syndromepatients (19 cases out of 47) were detected, and no (0) benign epilepsypatients were detected.

These results suggest that by examining both the SCN1A gene mutation andthe CACNA1A gene mutation, it is possible to eliminate the falsepositive (benign febrile seizure patients) better than examining justthe SCN1A gene mutation, and suggest a possibility of detecting theDravet syndrome patients with higher accuracy.

Example 3 Study of Gene Mutation in a Healthy Person

To investigate whether the remaining 6 kinds of gene mutations excludingthe registered 3 kinds out of the 9 kinds of missense mutations anddeletion mutations detected in the coding region of the CACNA1A gene areof the gene polymorphism (SNP), gene mutation of the CACNA1A gene wassimilarly analyzed for DNA extracted from blood of 190 healthy persons.Results of the 9 kinds of the missense mutations and deletion mutationsdetected in the coding region of the CACNA1A gene are shown in Table 6.As a result, one kind of the CACNA1A gene mutation (G266S) was notdetected from the healthy persons. From this result, it was found thatthe CACNA1A gene mutation of G266S is not an SNP, and is a novel genemutation (gene abnormality) not found in the 190 healthy persons, whichneither is in the NCBI SNP database.

TABLE 6 CACNA1A gene mutation detected in healthy persons and Dravetsyndrome Nucleotide Amino Acid Control Exon Substitution SubstitutionDravet (n = 47) (n = 188-190) p-value Frequency of variants  6 A876GG266S 1/47 2.1% 0/188   0% 0.20 11 A1415G K472R 1/47 2.1% 1/188 0.53%0.36 19 A2762C E921D 11/47  23.4%  49/188  26.06%  0.71 19 C2771G A924G1/47 2.1% 7/190 3.68% 1.00 19 A2987T E996V 11/47  23.4%  49/188  26.06% 0.71 20 G3322A G1108S 3/47 6.4% 16/189  8.46% 0.77 20 G3377A R1126H 4/47* 8.5% 1/188 0.53% 0.0061 47 G6602A R2201Q 4/47 8.5% 4/189 2.12%0.052 47 6605-6616del DQER2202- 1/47 2.1% 3/190 1.58% 1.00 2205delFrequency of combined mutations 19 E921D + E996V 11/47  23.4%  49/188 26.06%  0.71 20 + 47 R1126H + R2201Q  4/47* 8.50%  0/188   0% 0.0014

As a result of studying the comparison of frequencies in which mutationsoccur in healthy persons and Dravet syndrome patients, it was shown thatthe CACNA1A gene mutation R1126H was of a larger number with Dravetsyndrome in terms of statistical significance (p=0.0061), and it wasfound that the CACNA1A gene mutation R2201Q also had a trend having alarger number with Dravet syndrome patients (p=0.052). The patientssimultaneously having both mutations of R1126H and R2201Q on the CACNA1Agene were detected significantly in just the Dravet syndrome patients (4cases out of 47 cases), and no healthy persons were detected (p=0.0014).Examination of DNA of the parents of these four patients revealed thatthe two mutations of R1126H and R2201Q were simultaneously present onone chromosome, i.e. within the same CACNA1A protein molecule, and thatthis double mutation was inherited from the parents.

Example 4 Study of Relation Between Genotype and Symptoms

A study was performed on how the 9 kinds of missense mutations anddeletion mutations detected in the coding region of CACNA1A gene giveeffect on the worsening of symptoms of the disease. Out of Dravetsyndrome patients whose seizure symptom data is managed in detail, theseizure symptoms under the age of 1 were compared between 20 patientswho have just the SCN1A gene mutation and 19 patients who have amutation on both the SCN1A gene and the CACNA1A gene. A result thereofis shown in Table 7. Note that “GTC” in Table 7 is an abbreviation of ageneralized tonic-clonic seizure, and “CPS” is an abbreviation of acomplex partial seizure.

TABLE 7 Relation of symptoms under the age of 1 with genotype Total no.of Type of Seizures Seizure Total no. prolonged Hemi- Myoclonic onset of(>10 min) GTC CPS convulsion seizure Genotype N (months) seizuresseizures (%) (%) (%) (%) SCN1A 20 5.6 ± 0.3  10.2 ± 1.2 2.4 ± 0.4  95 4550  15 mutation + No CACNA1A variants SCN1A 19 4.6 ± 0.4* 10.7 ± 1.3 4.4± 0.7* 95 26 84* 11 mutation + CACNA1A variants GTC: generalizedtonic-clonc seizure. CPS: complex partial seizure *p < 0.05

It was found that the patients having a CACNA1A variant, as compared tothe patients having no CACNA1A variant, are (i) significantly quicker inseizure onset (p=0.049), (ii) significantly greater in the number oftimes prolonged seizures occur, which prolonged seizure is a convulsionseizure that continues for 10 or more minutes (p=0.019), and (iii)significantly higher in the frequency that a hemiconvulsion occurs(p=0.041). This indicates that when there is a variation of the CACNA1Agene including the polymorphism in addition to a SCN1A gene abnormality,there is a possibility that the symptom may worsen.

Example 5 Analysis on Functions of Mutant Voltage-Gated Calcium IonChannel

An analysis was performed on functions of a mutant calcium ion channeland a normal (wild-type) calcium ion channel, with use of culture cells.First, cDNA of a human CACNA1A gene (SEQ ID NO.: 4) was used to preparean expression vector having a mutant CACNA1A (double mutation of G266S;R1126H; R2201Q; deletion 2202-2205; double mutation of R1126H andR2201Q) gene. After obtaining DNA fragments including the mutated partsby PCR, regions of a normal cDNA corresponding to those fragments weresubstituted with those fragments, to prepare the mutant cDNA. As acontrol, an expression vector (pMO14×2-CACNA1A) having a normal(wild-type) CACNA1A gene was used.

Analysis was performed on functions of the mutant calcium ion channeland the normal calcium ion channel, with use of the culture cells. Aα-subunit type 1 of the voltage-gated calcium ion channel Ca_(V)2.1,which is a CACNA1A gene product, had been subjected to functionadjustment by the α2δ subunit and β4 subunit that similarly configurethe voltage-gated calcium ion channel Ca_(V)2.1. Hence, an expressionvector having a CACNA1A gene that encodes a α-subunit type 1, and anexpression vector having a human CACNB4 gene (GenBank accession No.U95020) (SEQ ID NO.: 151) encoding a P4 subunit and a rabbit α2δ gene(GenBank accession No. NM_(—)001082276) (SEQ ID NO.: 152) encoding a α2δsubunit. were coexpressed on a human renal cell HEK293 with use of atransfection reagent. Electrophysiologic properties were studied bypatch clamping of a whole cell record.

More specifically, recording of a calcium ion channel current wascarried out at room temperature of 22° C. to 24° C., 72 hours aftertransfection. With use of a multistage P-97 Flaming-Brown micropipettepuller, a patch electrode was prepared from borosilicate glass.

The composition of intracellular fluid was 110 mM CsOH, 20 mM CsCl, 5 mMMgCl₂, 10 mM EGTA, 5 mM MgATP, 5 mM creatine-phosphate, and 10 mM HEPES.On the other hand, the composition of the used extracellular fluid was 5mM BaCl, 150 mM TEA-C1, 10 mM glucose, and 10 mM HEPES. The amplifierused was Axopatch200B (Axon Instruments).

Electrophysiologic properties of the mutation channel were compared withthose of a normal channel, by studying voltage-gated channel activation,inactivation, recovery from inactivation, and duration current. Theactivation curve and the inactivation curve were analyzed by Boltzmannfunction, to find a half-maximal activation/inactivation (V_(1/2)) and aslope factor (k). The recovery curve from the inactivation was analyzedby a two exponential function. Statistics used the unpaired Student's ttest. Clampfit 8.2 software and OriginPro 7.0 (OriginLab) were used fordata analysis.

FIG. 13 and FIG. 14 are views illustrating results of performingfunction analysis of the calcium ion channel, by patch clamping. In thegraphs in FIG. 13 and FIG. 14, the normal calcium ion channel is shownas “WT”, and the mutant calcium ion channels are shown as “R266S”,“R1126H”, “R2201Q”, “De12202”, and “RH+RQ”. The mutation “De12202” meansthe mutation “Deletion 2202-2205”, and the mutation “RH+RQ” means themutation “R1126H+R2201Q”.

Illustrated in (a) of FIG. 13 is a barium current record in accordancewith a change in potential of the normal calcium ion channel and themutant calcium ion channel. Illustrated in (b) is a current-voltagerelationship, and illustrated in (c) are a peak current value (pA), atotal charge (pF), and a peak current density (pA/pF).

More specifically, (a) of FIG. 13 illustrates a current record ofmeasuring barium current that is depolarized by changing a depolarizingstimulus by 10 mV each from −40 mV to +60 mV and is flowed therein. Thecurrent-voltage relationship illustrated in (b) of FIG. 13 is a graphobtained by (i) measuring a flowing barium current for every membranepotential while having a holding potential, being deeper than a restingmembrane potential, as −100 mV, and a depolarizing stimulus beingchanged by 10 mV each from −40 mV to +60 mV, and (ii) plotting themembrane potential on a horizontal axis and a current value on avertical axis. The view illustrated on the lower right of the graph in(b) of FIG. 13 shows that in this experiment, “the depolarizing stimuluswas changed by 10 mV each from −40 mV to +60 mV for 30 ms(milliseconds), with the holding potential being −100 mV, which holdingpotential is deeper than the resting membrane potential”.

As a result, it was found that the mutant calcium ion channel“Deletion2202-2205” and “R1126H+R2201Q” significantly increased in itsflowed current amount, peak current value, and peak current density, ascompared to the normal calcium ion channel.

Next, in order to specifically study the electrophysiologic propertiesof the calcium ion channel, a voltage-gated activity of the calcium ionchannel ((a) of FIG. 14), a time constant (τ) at activation ((b) and (c)of FIG. 14), inactivation of the calcium ion channel ((d) of FIG. 14),and a time constant (τ) at inactivation ((e) FIG. 14) were measured.

The activation curve illustrated in (a) of FIG. 14 shows a bariumcurrent value flowing per membrane potential as a relative value, byhaving a maximum sodium current value obtained from the graph of (b) ofFIG. 13 be 1, and an obtained curve was analyzed by Boltzmann functionto find a half-maximal activation (V_(1/2)) and a slope factor (k). Theview provided on the lower right of the graph in (a) of FIG. 14represents that, in this experiment, “the depolarizing stimulus waschanged by 10 mV each from −40 mV to +60 mV for 30 ms (milliseconds),with the holding potential being −100 mV, which holding potential isdeeper than the resting membrane potential”.

As a result of analyzing the voltage-gated activity of the calcium ionchannel, it was found that (i) the mutant calcium ion channel “G266S”and “R1126H” show a significant hyperpolarization shift as compared tothe normal channel, and that (ii) the mutant calcium ion channel“R1126H” and “Deletion2202-2205” significantly increased in thevoltage-gated property as compared to the normal channel, by comparingthe slope factor (k) (see (a) of FIG. 14 and Table 8). This means thatthe mutant calcium ion channel “G266S”, “R1126H” and “Deletion2202-2205”are easily activated even in a low membrane potential, thereby tendingto cause excess hyperexcitability of nerve cells.

Table 8 shows electrophysiologic properties of the calcium ion channel.Statistical comparison of the normal CACNA1A and the mutant CACNA1A wereperformed by the Student's t test. The asterisk (*) in Table 8 indicatesthat there is a significant difference between the normal CACNA1A andthe mutant CACNA1A when a critical rate is under 5%, and the doubleasterisk (**) indicates that there is a significant difference betweenthe normal CACNA1A and the mutant CACNA1A when the critical rate isunder 1%.

TABLE 8 Electrophysiologic properties of calcium ion channel ActivationV_(1/2) Inactivation (mV) k (mV) n V_(1/2) (mV) k (mV) n WT- 6.3 ± 4.3 ±0.2 16 −16.9 ± 1.5 −4.5 ± 0.6 10 CACNA1A 1.3 G266S 1.0 ± 4.3 ± 0.4 11−13.8 ± 1.6 −5.5 ± 0.3 10 1.2** R1126H 0.4 ± 3.3 ± 0.3* 10 −18.9 ± 0.6−6.1 ± 0.7 8 1.6** R2201Q 6.4 ± 4.1 ± 0.2 8 −13.4 ± 1.7 −5.7 ± 0.4 101.5 Deletion2202- 1.3 ± 3.4 ± 0.2* 8 −13.3 ± 1.2 −4.7 ± 0.6 9 2205 1.4R1126H + 2.6 ± 3.5 ± 0.2 10 −15.2 ± 0.9 −5.4 ± 0.1 10 R2201Q 1.1V_(1/2), half-maximal voltage activation and inactivation; k, slopefactor. Statistical coparison between WT-CACNA1A and mutant channels wasperformed by Student's t test (*P < 0.05 and **P < 0.01 versusWT-CACNA1A).

Illustrated in (b) of FIG. 14 is a time constant of channelvoltage-gated activation, that is to say, a time required for eachcurrent to reach 66.7%. Moreover, (c) of FIG. 14 illustrates a timeconstant of voltage-gated activation at 20 mV. From (b) and (c) of FIG.14, it was demonstrated that the mutant calcium ion channel “G266S” wassignificantly small in the time constant of voltage-gated activation at20 mV, as compared to a normal channel. Since this point is consideredas that the mutant calcium ion channel “G2665” is made so as to flow alot of current within a short depolarization, this means that there is atrend of causing hyperexcitement in the nerve cells.

Illustrated in (d) of FIG. 14 is a voltage-gated inactivation curve ofthe calcium ion channel, which was measured upon changing a membranepotential to activate the calcium ion channel and thereafter providing adepolarizing stimulus to measure how much barium current was flown. Notethat the view illustrated on the lower left of the graph illustrated in(d) of FIG. 14 shows that, in this experiment, “the depolarizingstimulus was changed by 20 mV each from −120 mV to +60 mV for 2 s(seconds), and subsequently be changed to 20 mV, with the holdingpotential being −100 mV, which holding potential is deeper than theresting membrane potential”.

The voltage-gated inactivation curve of the calcium ion channel showedno recognizable significant difference, in either of the mutant channelor the normal channel.

Illustrated in (e) of FIG. 14 is a result of studying an inactivationtime constant (τ). There are two kinds of inactivation: inactivation ofa fast component and inactivation of a slow component. The “τ_(fast)” inthe left graph of (e) of FIG. 14 is a constant representing a timerequired until the inactivation of the fast component reaches 33.3%, andthe “τ_(slow)” in the right graph is a constant representing a timerequired until the inactivation of the slow component reaches 33.3%.These inactivation time constants were, more specifically, calculated byanalyzing the inactivation curve with use of Clampfit 8.2 software.

As a result, there was no significant difference in the inactivationtime constant between that of the normal calcium ion channel and that ofthe mutant calcium ion channel. Table 9 shows physiological propertiesof the mutant calcium ion channel. The arrow pointing upwards (↑) inTable 9 indicates that an increase in channel activity was recognized,and the hyphen “-” indicates that no change was recognized in thechannel activity.

TABLE 9 Summary of electrophysiological properties of mutant calcium ionchannel CACNA1A Biophysical Del R1126H + property G266S R1126H R2201Q2202-2205 R2201Q Peak current density — — — ↑ ↑↑ Activation V_(1/2) ↑ ↑— — — Activation slop — ↑ — ↑ — factor Activation time ↑ — — — —constants Inactivation V_(1/2) — — — — — Inactivation slope — — — — —factor ↑, predicted gain of channel activity. —, no predicted change inchannel activity.

It was found that the mutations other than “R2201Q” in the calcium ionchannel were mutations of a gain of function kind, and tends to causeexcitement of the nerve cells.

Example 6 Production of Dravet Syndrome Model Rat

From the foregoing findings, it was considered that having some kind ofmutation on both of SCN1A and CACNA1A is important in the development ofDravet syndrome. Accordingly, a rat was produced which has both of themutation on α1-subunit gene Scn1a of the voltage-gated sodium ionchannel Na_(V)1.1 and the mutation on α1-subunit gene Cacna1a of thevoltage-gated calcium ion channel Ca_(V)2.1, to study the worsening ofsymptoms (human genes are represented as SCN1A and CACNA1A, and ratgenes are represented as Scn1a and Cacna1a).

More specifically, a rat having a mutation on the Scn1a gene(F344-Scn1a^(Kyo811)) and a rat having a mutation on the Cacna1a gene(GRY (groggy rat, Cacna1a^(gry))) were used as parent rats. Each ofthese mice is described below.

<F344-Scn1a^(Kyo811)>

A rat produced by ENU mutagenesis, having a missense mutation on a α1subunit gene (Scn1a) of the voltage-gated sodium channel Na_(V)1.1.Asparagine (N), which is an amino acid at position 1417, was mutated tohistidine (H) (represented as “N1417H”). This rat served as a modelanimal of human generalized epilepsy febrile seizure plus (GEFS+).Background genealogy is F344/NS1c rat. This rat was provided from theInstitute of Laboratory Animals, Graduate School of Medicine, KyotoUniversity.

<GRY (Groggy Rat, Cacna1a^(gry))>

A mutant rat produced by administering methyl nitrosourea to Scl:Wistar,whose main symptoms are ataxia and absence-like seizure. This rat has anautosomal recessive mode of inheritance, and has a missense mutation onthe α1-subunit of the voltage-gated calcium ion channel Ca_(V)2.1.Methionine (M), which is an amino acid at position 251, is mutated tolysine (K) (M251K). This rat was provided from the Institute ofLaboratory Animals, Graduate School of Medicine, Kyoto University.

FIG. 11 is a view showing an amino acid sequence of a protein encoded bya human CACNA1A gene and an amino acid sequence of a protein encoded bya rat Cacna1a gene. The upper line of the amino acid sequence shown inFIG. 11 represents an amino acid sequence of the protein encoded by therat Cacna1a gene (GenBank accession No. NM_(—)012918) (SEQ ID NO.: 147),and the lower line is the amino acid sequence of the protein encoded bythe human CACNA1A gene (GenBank accession No. NM_(—)023035) (SEQ ID NO.:3). Moreover, the squared amino acid “M” in FIG. 11 is an amino acidthat is mutated from the amino acid “M” to an amino acid “K” in thehuman mutant CACNA1A (M249K) protein (SEQ ID NO.: 148) and the ratmutant Cacna1a (M251K) protein (SEQ ID NO.: 149).

As illustrated in FIG. 11, the mutation (M251K) on the α1 subunit of therat voltage-gated calcium ion channel Ca_(V)2.1 corresponds to themutation (M249K) on the al subunit of the human voltage-gated calciumion channel Ca_(V)2.1.

The F344-Scn1a^(Kyo811) and GRY (groggy rat, Cacna1a^(gry)) as describedabove were mated to produce a rat having each of the gene mutations.

(1. Analysis on Functions of Mutant Voltage-Gated Sodium Ion Channel)

An analysis was performed with use of culture cells, on functions of amutant sodium ion channel and normal sodium ion channel, before testsusing the rats were performed. The rat having a mutation on the Scn1agene (F344-Scn1a^(Kyo811)) has asparagine (AAT), which is an amino acidat position 1417 of a protein encoded by the Scn1a gene, was changed tohistidine (CAT) (N1417H). The asparagine at position 1417 is located ina pore formation region that is related to ionic permeation of sodiumion channel third domain. On this account, first, the function analysisof the mutant voltage-gated sodium ion channel included inF344-Scn1a^(Kyo811) was performed.

More specifically, an expression vector having a mutant SCN1A (N1417H)gene (SEQ ID NO.: 150) including a missense mutation was prepared withuse of cDNA of human SCN1A gene. As control, an expression vector havinga normal (wild-type) SCN1A gene (SEQ ID NO.: 2) was prepared.

FIG. 1 is a view showing an amino acid sequence of a protein encoded bythe human SCN1A gene and an amino acid sequence of a protein encoded bythe rat Scn1a gene. The upper line in the amino acid sequence shown inFIG. 1 represents an amino acid sequence of a protein that is encoded bythe human SCN1A gene (SEQ ID NO.: 1), and the lower line represents anamino acid sequence of a protein that is encoded by the rat Scn1a gene(SEQ ID NO.: 144). Moreover, the squared amino acid “N” in FIG. 1 is anamino acid on which a mutation from an amino acid “N” to an amino acid“H” occurs, of the human mutant SCN1A (N1417H) protein (SEQ ID NO.: 145)and the rat mutant SCN1A (N1417H) protein (SEQ ID NO.: 146).

An analysis was performed with use of culture cells, on functions of themutant sodium ion channel and the normal sodium ion channel. Theα-subunit type 1 of the voltage-gated sodium ion channel Na_(V)1.1,which is a SCN1A gene product, was adjusted in its function by β₁subunit and β₂ subunit that similarly make up the voltage-gated sodiumion channel Na_(V)1.1. Hence, an expression vector having the SCN1A genethat encodes the α-subunit type 1 was coexpressed with an expressionvector having the SCN1B gene that encodes the β₁ subunit and the SCN2Bgene that encodes the β₂ subunit in a human renal cell HEK293, with useof a transfection reagent. The electrophysiologic properties werestudied by patch clamping based on whole cell recording.

More specifically, recording of the sodium ion channel current wascarried out at room temperature of 22° C. to 24° C., 24 hours to 48hours after transfection. A patch electrode was prepared fromborosilicate glass by use of multistage P-97 Flaming-Brown micropipettepuller.

Composition of intracellular fluid was 110 mM CsF, 10 mM NaF, 20 mMCsCl, 2 mM EGTA, and 10 mM HEPES. On the other hand, the composition ofextracellular fluid was 145 mM NaCl, 4 mM KCl, 1.8 mM CaCl₂, 1 mM MgCl₂,and 10 mM HEPES. Axopatch200B (Axon Instruments) was used as theamplifier.

Electrophysiologic properties of the mutation channel were compared withthose of a normal channel, by studying voltage-gated channel activation,inactivation, recovery from inactivation, and duration current. Theactivation curve and the inactivation curve were analyzed by Boltzmannfunction, to find a half-maximal activation/inactivation (V_(1/2)) and aslope factor (k). The recovery curve from the inactivation was analyzedby a two exponential function. Durable Na current was found by adifference in the duration current when depolarized at −10 mV for 100ms, before and after addition of 10 μM of tetrodotoxin (TTX). Statisticsused were unpaired Student's t test. Clampfit 8.2 software and OriginPro7.0 (OriginLab) were used for data analysis.

FIGS. 2 to 4 are views illustrating results of performing functionanalysis of the sodium ion channel by patch clamping. The graphs ofFIGS. 2 to 4 show the normal sodium ion channel as “WT” or “WT-SCN1A”,and show the mutant sodium ion channel as “N1417H”.

Illustrated in (a) of FIG. 2 is a typical example of a sodium current inresponse to a change in potential of the normal sodium ion channel andthe mutant sodium ion channel. More specifically, a depolarizingstimulus was changed 10 mV each from −80 mV to +60 mV fordepolarization, and sodium current that flowed in was measured. As aresult, both of the normal sodium ion channel and the mutant sodium ionchannel function as a channel, and there was no significant differencebetween the two.

Illustrated in (b) of FIG. 2 is a result of studying the inactivationtime constant (τ). There are two types of inactivation; an inactivationof a fast component and an inactivation of a slow component. The “τ1” in(b) of FIG. 2 is indicative of a constant indicative of a time requiredfor the inactivation of the fast component to reach 33.3%, and the “τ2”is indicative of a constant indicative of a time required for theinactivation of the slow component to reach 33.3%. These inactivationtime constants, more specifically, were calculated by analyzing theinactive curve with use of the Clampfit 8.2 software. As a result, therewas no significant difference in the inactivation time constant betweenthat of the normal sodium ion channel and that of the mutant sodium ionchannel.

Next, in order to specifically study the electrophysiologic propertiesof the sodium ion channel, a current-voltage relationship ((a) of FIG.3), an activation of the sodium ion channel ((b) of FIG. 3), aninactivation of the sodium ion channel ((c) of FIG. 3), and recoveryfrom the inactivation of the sodium ion channel ((d) of FIG. 3) weremeasured.

More specifically, the current-voltage relationship illustrated in (a)of FIG. 3 was obtained by (i) measuring a flowing sodium current forevery membrane potential while having a holding potential, being deeperthan a resting membrane potential, as −120 mV, and a depolarizingstimulus being changed by 10 mV each from −80 mV to +60 mV, and (ii)plotting the membrane potential on a horizontal axis and a current valueon a vertical axis. The view illustrated on the lower left of the graphin (a) of FIG. 3 shows that in this experiment, “the depolarizingstimulus was changed by 10 mV each from −80 mV to +60 mV for 20 ms(milliseconds), with the holding potential being −120 mV, which holdingpotential is deeper than the resting membrane potential”.

The activation curve illustrated in (b) of FIG. 3 shows a sodium currentvalue flowing per membrane potential as a relative value, by having amaximum sodium current value obtained from the graph of (a) of FIG. 3 be1, and an obtained curve was analyzed by Boltzmann function to find ahalf-maximal activation (V_(1/2)) and a slope factor (k). The viewprovided on the lower right of the graph in (b) of FIG. 3 representsthat in this experiment, “the depolarizing stimulus was changed by 10 mVeach from −80 mV to +60 mV, for 20 ms (milliseconds), with the holdingpotential being −120 mV, which holding potential is deeper than theresting membrane potential”.

The inactive curve illustrated in (c) of FIG. 3 was obtained bysimilarly changing the membrane potential to activate the channel andthereafter providing depolarizing stimulus and measuring how much thesodium current flows, to find the half-maximal inactivation (V_(1/2))and the slope factor (k). Note that the view provided on the lower leftof the graph of (c) of FIG. 3 represents that in this experiment, “thedepolarizing stimulus was changed by 10 mV each from −140 mV to +0 mVfor 100 ms (milliseconds) and subsequently changed to −10 mV, with theholding potential being −120 mV”.

The recovery curve from the inactivation illustrated in (d) of FIG. 3was obtained as follows. When a depolarizing stimulus was provided withpulse 1 (P1), the channel became inactive upon opening. When thedepolarizing stimulus was returned to the original −120 mV, the sodiumion channel returned to its resting state, and upon stimulation of pulse2 (P2), the channel opened again. The recovery time of this pulse 1 andpulse 2 were changed to obtain the recovery curve from the inactivation.This curve was analyzed by a two exponential function. It was determinedwhether the function of the channel was made easily excited or in theopposite was made difficult to be excited, depending on whether therecovery was quicker or slower as compared to the normal channel. Theview provided on the lower right of the graph of (d) of FIG. 3 indicatesthat in this experiment, “a holding potential was mV, −10 mV wasprovided for 100 ms (milliseconds) as the depolarizing stimulus andthereafter was returned to −120 mV, and after elapse of each of thetimes (milliseconds) shown on the x-axis, −10 mV was provided for 20 ms(milliseconds)”.

As a result, no significant difference was recognized in thecurrent-voltage relationship and the channel activation, between thenormal sodium ion channel and the mutant sodium ion channel (see (a) and(b) of FIG. 3). Meanwhile, a significant test was performed regardingthe channel inactivation, on a point that the normal sodium ion channeland the mutant sodium ion channel are inactivated by 50%, wherebyresulted in finding that the mutant sodium ion channel had shiftedsignificantly to the depolarization side (p<0.05) ((c) of FIG. 3).

As to the recovery from the channel inactivation, it was found that therecovery was significantly slow in the mutant sodium ion channel ((d) ofFIG. 3). In (d) of FIG. 3, a part in which a period of recovery(Recovery period (ms)) from the inactivation was 1 ms to 8 mscorresponds to a “fast component”, and a part in which the period ofrecovery from the inactivation was 10 ms to 100 ms corresponds to a“slow component”.

More specifically, upon comparison between the normal sodium ion channeland an abnormal sodium ion channel based on a time required for the fastcomponent in recovering from the inactivation to recover from theinactivation to 33.3%, it was found that the recovery was significantlyslow for the mutant sodium ion channel (normal: τ_(f)=1.7±0.1 ms, n=14;mutant: τ_(f)=2.5±0.2 ms (P<0.01), n=12).

Similarly, upon comparison of the normal sodium ion channel with theabnormal sodium ion channel based on the time required for the slowcomponent in recovering from the inactivation to recover from theinactivation to 33.3%, it was found that the mutant sodium ion channelwas significantly slow in recovering (normal: τ_(f)=40.3±5.3 ms, n=14;mutant: τ_(s)=60.9±7.9 ms (P<0.05), n=12).

FIG. 4 shows that, even if the sodium ion channel was made inactivatedafter the potential was changed to activate the sodium ion channel, thebaseline of the mutant sodium channel does not return back in the wholecell record, which indicates clearly that the sodium current waspersistently flowing into the mutant sodium ion channel. The persistentsodium current is considered as an obstruction of an inactivation gate.From the view of (a) of FIG. 4, it was confirmed that even after theelapse of time, the inactivation was insufficient in the mutant sodiumion channel as compared to that of the normal sodium ion channel.

So as to find the persistent sodium current shown in (a) of FIG. 4, arelative value (%) was found by dividing, with a maximum current amount,a final current amount that flowed between 80 milliseconds to 100milliseconds when a depolarizing stimulus of 100 milliseconds was given.Results thereof are shown in (b) of FIG. 4. From these results, it wasfound that the mutant sodium ion channel had properties that thepersistent sodium current increases.

This data show that the function of the voltage-gated sodium ion channelNa_(V)1.1 became abnormal by the mutation. Namely, this means that byhaving the mutation, the nerve cells are easily excessively excited,that is to say, more easily causes the occurrence of a convulsion.

Literature (Satoko Tokuda et. al., BRAINRESEARCH 1133 (2007) 168-177;Kenta Tanaka et. al., Neuroscience Letters 426 (2007) 75-80) disclosesthat the function of the voltage-gated calcium ion channel Ca_(V)2.1 ofa rat becomes abnormal due to a mutation (M251K) on the α1 subunit ofthe voltage-gated calcium ion channel Ca_(V)2.1 of the rat.

Therefore, with a rat having the mutation on both the Scn1a gene andCacna1a gene described later, it can be considered that the functions ofboth the voltage-gated sodium ion channel Na_(V)1.1 and thevoltage-gated calcium ion channel Ca_(V)2.1 are abnormal.

(2. Confirmation of Gene Mutation in Dravet Syndrome Model Rat)

The foregoing F344-Scn1a^(Kyo81) and the GRY (groggy rat, Cacna1a^(gry))were mated as parent rats (P) to produce F1 (first filial generation)rats, and these F1 rats were mated to produce F2 (second filialgeneration) rats. FIG. 5 is a view showing genotypes of the parent rats(P), the F1 rats and the F2 rats. As illustrated in (a) of FIG. 5, theF1 rats have the heterozygous mutation on both the Scn1a gene and theCacna1a gene (referred to as “Scn1a mutant (hetero)+Cacna1a mutant(hetero)”). Moreover, as illustrated in (b) of F1G. 5, rats showing 9types of genotypes were born from the F2 rats. The genotypes of each ofthe rats were identified by extracting a tip tissue of the tail of therats and extracting its DNA, to perform DNA sequencing with theextracted DNA and detect its gene mutation, or by detecting a digestedpattern with use of a restriction enzyme.

(Method of Confirming Gene Mutation by DNA Sequencing)

Confirmation of gene mutation by DNA sequencing was performed asfollows. First, a genomic DNA was amplified with use of a primer pairthat sandwiches a mutation point (a nucleotide sequence of a Scn1aamplification primer pair is represented by SEQ ID NO.: 5 and SEQ IDNO.: 6, and a nucleotide sequence of a Cacna1a amplification primer pairis represented by SEQ ID NO.: 7 and SEQ ID NO.: 8), and thereafter, anobtained PCR product was purified with use of a PCR productspre-sequencing kit (Amersham Biosciences, Little Chalfont,Buckinghamshire, England). See the item “Sequence of primers” laterdescribed for the nucleotide sequence of the used primer pairs.

Next, sequence reaction was performed with use of a Big Dye TerminatorFS ready-reaction kit (Applied Biosystems), to determine a nucleotidesequence with a fluorescence sequencer (ABI PRISM3100 sequencer; AppliedBiosystems).

FIG. 6 is a view illustrating a method of identifying a genotype of theScn1a gene and the Cacna1a gene of the F2 rats, by sequencing. Asillustrated in FIG. 6, a wild-type Scn1a gene has a nucleotide atposition 4249 be “A”. In comparison, a mutant Scn1a gene (N1417H) has anucleotide at position 4249 that is mutated from “A” to “C”. As aresult, a codon “AAT” that designates asparagine (N) being an amino acidat position 1417 in the wild-type Scn1a gene, is mutated to a codon“CAT” which designates histidine (H), in the mutant Scn1a gene (N1417H).

Moreover, the wild-type Cacna1a gene has a nucleotide at position 752 be“T”. In comparison, the mutant Cacna1a gene (M251K) has a nucleotide atposition 752 that is mutated from “T” to “A”. As a result, a codon “ATG”that designates methionine, which is an amino acid at position 251, ismutated to a codon “AAG” that designates lysine.

(Method of Confirming Gene Mutation by Restriction Enzyme Digestion)

The method of confirming gene mutation by the restriction enzymedigestion was performed as follows. When detecting mutation in the Scn1agene, a genomic DNA was amplified with use of a primer pair (SEQ IDNOs.: 5 and 6) that sandwich a mutation point in the Scn1a gene, andthereafter an obtained PCR product was reacted for three hours at 50°C., with use of a restriction enzyme BclI. Thereafter, the PCR productreacted with the restriction enzyme was subjected to electrophoresiswith use of 4% agarose gel, and the size of the band was detected. FIG.7 is a view illustrating a method of identifying the genotype of theScn1a gene of the F2 rats, by restriction enzyme digestion.

As shown in (a) and (b) of FIG. 7, the wild-type Scn1a gene was notdigested with BM so the size of the band remained as the size of the PCRproduct (nucleotide of 380 bp). On the other hand, the mutant Scn1a gene(N1417H) was digested with BM so two fragments (nucleotides of 276 bpand 104 bp) were detected. In a case of a heterozygous rat of thewild-type Scn1a gene and the mutant Scn1a gene (N1417H), three fragments(nucleotides of 380 bp, 276 bp, and 104 bp) were detected. Illustratedin (c) of FIG. 7 shows a result of electrophoresis.

In a case of detecting the mutation on the Cacna1a gene, a genomic DNAwas amplified with use of a primer pair (SEQ ID NOs.: 7 and 8) thatsandwich a mutation point of the Cacna1a gene, and thereafter, anobtained PCR product was reacted for hour at 37° C. with use of arestriction enzyme PciI. Thereafter, the PCR product reacted with therestriction enzyme was subjected to electrophoresis with use of 4%agarose gel, to detect the size of a band.

FIG. 8 is a view illustrating a method of identifying a genotype of theCacna1a gene of the F2 rats, by restriction enzyme digestion. Asillustrated in (a) and (b) of FIG. 8, a wild-type Cacna1a gene was notdigested with PciI, so hence the size of the band remained as the sizeof the PCR product (nucleotide of 352 bp). On the other hand, the mutantCacna1a gene (M251K) was digested with PciI, and thus two fragments(nucleotides of 219 by and 133 bp) were detected. With a heterozygousrat of the wild-type Cacna1a gene and an abnormal Cacna1a gene (M251K),three fragments (nucleotides of 352 bp, 219 bp, and 133 bp) weredetected. Illustrated in (c) of FIG. 8 is a result of electrophoresis.

Example 7 Analysis of Dravet Syndrome Model Rat

A study was performed on what kind of (worsening) effect was given onthe seizure when a mutation on the Cacna1a gene was added to a mutationon the Scn1a gene, with use of a Dravet syndrome model rat. Morespecifically, comparison was made regarding symptoms when a convulsionseizure was induced by heat load, between a rat having a homozygousmutation on the Scn1a gene (referred to as “Scn1a mutant (homo)+Cacna1awild-type (homo)”) and a rat having a homozygous mutation on the Scn1agene and a heterozygous mutation on the Cacna1a gene (referred to as“Scn1a mutant (homo)+Cacna1a mutant (hetero)”).

The Scn1a mutant (homo)+Cacna1a wild-type (homo) and the Scn1a mutant(homo)+Cacna1a mutant (hetero) both have a homozygous mutation on theScn1a gene (N1417H). Hence, comparison is made between the wild-typeCacna1a gene and the mutant Cacna1a gene (M251K), under the condition ofthe homozygous mutation of the Scn1a gene.

Moreover, a rat having a wild-type Scn1a gene and a wild-type Cacna1agene (referred to as “Scn1a wild-type (homo)+Cacna1a wild-type (homo)”)and a rat having a wild-type homozygous mutation on the Scn1a gene and aheterozygous mutation on the Cacna1a gene (referred to as “Scn1awild-type (homo)+Cacna1a mutation (hetero)”) were used as control. Thefollowing lists the genotypes of the rats used in the experiment. Thefollowing numbers (1) to (4) correspond to the numbers in (b) of FIG. 5.

(1) Scn1a^(wt)/^(wt)Cacna1a^(wt)/^(wt) (Scn1a wild-type (homo)+Cacna1awild-type (homo)) 14 males

(2) Scn1a^(mut)/^(mut) Cacna1a^(wt)/^(wt) (Scn1a mutant (homo)+Cacna1awild-type (homo)) 7 males

(3) Scn1a^(mut)/^(mut) Cacna1a^(wt)/^(mut) (Scn1a mutant (homo)+Cacna1amutant (hetero)) 17 males

(4) Scn1a^(wt)/^(wt) Cacna1a^(wt)/^(mut) (Scn1a wild-type (homo)+Cacna1amutant (hetero)) 12 males.

Hot bath load (45° C.) were given on male rats of 5 weeks old of thegroups (1) to (4) described above, to compare their body temperatures ata time when a convulsion is induced, their duration of the convulsion,and their severity score of the convulsion. A rectal temperature at thetime when the seizure started was measured, to serve as the bodytemperature at the time when the convulsion was induced. The seizureseverity score of the convulsion were evaluated as follows: 0=noseizure, 1=facial convulsion, 2=clonic convulsion of both arms whilemaintaining posture, 3=sprint or jump, 4=generalized convulsion unableto maintain posture, and 5=death caused by persistent convulsion.

The results were as shown in FIG. 9. FIG. 9 is a view showing a resultof the effect caused by the mutation on the Cacna1a gene in the Scn1agene-mutated rat. In the graphs of (a) to (c) in FIG. 9,Scn1a^(mut)/^(mut)Cacna1a^(wt)/^(wt) (the foregoing rat (2)) is shown as“Scn1a mutant (homo)”. Scn1a^(mut)/^(mut)Cacna1a^(wt)/^(mut) (theforegoing rat (3)) is shown as “Scn1a mutant (homo)+Cacna1a mutant(hetero)”. Moreover, control Scn1a^(wt)/^(wt)Cacna1a^(wt)/^(wt)(foregoing rat (1)) is shown as “WT”, and controlScn1a^(wt)/^(wt)Cacna1a^(wt)/^(mut) (foregoing rat (4)) is shown as“Cacna1a mutant (hetero)”.

As a result of analysis, the group (3) rats (Scn1a mutant (homo)+Cacna1amutant (hetero)) had no large difference in the body temperatures at thetime of convulsion onset (convulsion threshold) ((a) of FIG. 9) andseverity scores ((b) of FIG. 9), from those of the group (2) rats (Scn1amutant (homo)+Cacna1a wild-type (homo)). However, it was found that theduration of the convulsion ((c) of FIG. 9) became significantly long.This result demonstrates that the mutation of the Cacna1a gene relatesto the worsening of the symptoms of convulsion.

Furthermore, FIG. 10 shows a part of an electroencephalogram during aseizure of a group (3) rat (Scn1a mutant (homo)+Cacna1a mutant(hetero)). It was considered from this result that a rat having amutation on the Scn1a gene and the Cacna1a gene could serve as a modelrat of the intractable Dravet syndrome. The model rat is expected to beusefully used in the future for clarification of the onset mechanism ofthe intractable Dravet syndrome, development of medicament for Dravetsyndrome, and like uses.

Moreover, these results are considered as supporting the gene analysisdata of Example 1, that a variation of the CACNA1A gene was detected inaddition to a mutation on the SCN1A gene, in a patient of Dravetsyndrome which is an intractable epilepsy. Namely, the method accordingto the present invention of obtaining data for assessing the potentialfor development of Dravet syndrome can be said as a technique supportedby the gene analysis results of the Examples, a mutant channel functionanalysis result, and animal experiment results.

CONCLUSION

The present invention was developed based on a molecular foundation ofdevelopment of the intractable Dravet syndrome; the assessment methodaccording to the present invention can be said as useful as an earlydetection method of Dravet syndrome patients. By use of the assessmentmethod according to the present invention, it is possible to find Dravetsyndrome, which has an unfavorable prognosis, in high accuracy and at anearly stage. This allows for an epilepsy specialist to prepare atreatment management system for the patient of Dravet syndrome from anearly stage. As a result, this leads to improvement in therapeuticintervention of the patient, reduction of mental load on the family, andreduction of economical burden. Moreover, it is possible to carry outappropriate treatment to the Dravet syndrome patient, so therefore isconsidered as contributive to the reduction of medical fees.

Furthermore, with use of the kit according to the present invention, itis possible to easily detect the mutation for both the SCN1A gene andCACNA1A gene. Consequently, the kit according to the present inventionis useful for a general pediatrician to distinguish a patient of Dravetsyndrome who requires treatment by a specialist out of the benignfebrile epilepsies, during the initial stage of the disease under theage of one.

By use of the assessment method and the kit according to the presentinvention, it is possible to detect with high accuracy a patient ofDravet syndrome at the point in time of under the age of one, which wasdifficult to detect until now. Moreover, by examining gene abnormalitiesupon sending the blood taken to an examination center, it is possible todetect Dravet syndrome patients in high accuracy even for a remotepersonal hospital or the like.

Moreover, the model animal and cell according to the present inventionmay be usefully used in the clarification of an onset mechanism of theintractable Dravet syndrome, the development of medicament for Dravetsyndrome, and like uses.

<Primer Sequences>

Table 10 shows a nucleotide sequence of a primer pair used foramplifying the Scn1a gene and amplifying the Cacna1a gene.

TABLE 10  Scn1a Sense 5′-TGA CTT TTC TTT CTC TCC GTT TG-3′ SEQ IDamplification primer: NO.: 5 Antisense5′-TGG CTG CAA TAA TCA CTT TGT T-3′ SEQ ID primer: NO.: 6 Cacna1a Sense5′-TCT CTG TCT CCC CAG GTT TAC-3′ SEQ ID amplification primer: NO: 7Antisense 5′-GTG GCT AAC ACA CAG CTT TGC-3′ SEQ ID primer: NO.: 8

Tables 11 and 12 show nucleotide sequences of primer pairs used fordetecting SCN1A gene genomes.

TABLE 11  Exon 1 Sense 5′-tcatggcacagttcctgtatc-3′ SEQ ID amplificationprimer: NO.: 9 Antisense 5′-gcagtaggcaattagcagcaa-3′ SEQ ID primer:NO.: 10 Exon 2 Sense 5′-tggggcactttagaaattgtg-3′ SEQ ID amplificationprimer: NO.: 11 Antisense 5′-tgacaaagatgcaaaatgagag-3′ SEQ ID primer:NO.: 12 Exon 3 Sense 5′-gcagtttgggcttttcaatg-3′ SEQ ID amplificationprimer: NO.: 13 Antisense 5′-tgagcattgtcctcttgctg-3′ SEQ ID primer:NO.: 14 Exon 4 Sense 5′-agggctacgtttcatttgtatg-3′ SEQ ID amplificationprimer: NO.: 15 Antisense 5′-tgtgctaaattgaaatccagag-3′ SEQ ID primer:NO.: 16 Exon 5 Sense 5′-CAGCTCTTCGCACTTTCAGA-3′ SEQ ID amplificationprimer: NO.: 17 Antisense 5′-TCAAGCAGAGAAGGATGCTGA-3′ SEQ ID primer:NO.: 18 Exon 6 Sense 5′-agcgttgcaaacattcttgg-3′ SEQ ID amplificationprimer: NO.: 19 Antisense 5′-gggatatccagcccctcaag-3′ SEQ ID primer:NO.: 20 Exon 7 Sense 5′-gacaaatacttgtgcctttgaatg-3′ SEQ ID amplificationprimer: NO.: 21 Antisense 5′-acataatctcatactttatcaaaaacc-3′ SEQ IDprimer: NO.: 22 Exon 8 Sense 5′-gaaatggaggtgttgaaaatgc-3′ SEQ IDamplification primer: NO.: 23 Antisense 5′-aatccttggcatcactctgc-3′SEQ ID primer: NO.: 24 Exon 9 Sense  5′-agtacagggtgctatgaccaac-3′ SEQ IDamplification primer: NO.: 25 Antisense 5′-tcctcatacaaccacctgctc-3′SEQ ID primer: NO.: 26 Exon 10 Sense  5′-tctccaaaagccttcattagg-3′ SEQ IDamplification primer: NO.: 27 Antisense 5′-ttctaattctccccctctctcc-3′SEQ ID primer: NO.: 28 Exon 11 Sense  5′-tcctcattctttaatcccaagg-3′SEQ ID amplification primer: NO.: 29 Antisense5′-gccgttctgtagaaacactgg-3′ SEQ ID primer: NO.: 30 Exon 12 Sense 5′-gtcagaaatatctgccatcacc-3′ SEQ ID amplification primer: NO.: 31Antisense 5′-gaatgcactattcccaactcac-3′ SEQ ID primer: NO.: 32 Exon 13Sense  5′-tgggctctatgtgtgtgtctg-3′ SEQ ID amplification primer: NO.: 33Antisense 5′-ggaagcatgaaggatggttg-3′ SEQ ID primer: NO.: 34 Exon 14Sense  5′-tacttcgcgtttccacaagg-3′ SEQ ID amplification primer: NO.: 35Antisense 5′-gctatgcaagaaccctgattg-3′ SEQ ID primer: NO.: 36

TABLE 12  Exon 15 Sense  5′-atgagcctgagacggttagg-3′ SEQ ID amplificationprimer: NO.: 37 Antisense 5′-atacatgtgccatgctggtg-3′ SEQ ID primer:NO.: 38 Exon 16 Sense 5′-tgctgtggtgtttccttctc-3′ SEQ ID amplificationprimer: NO.: 39 Antisense 5′-tgtattcataccttcccacacc-3′ SEQ ID primer:NO.: 40 Exon 17 Sense 5′-aaaagggttagcacagacaatg-3′ SEQ ID  amplificationprimer:  NO.: 41 Antisense 5′-attgggcagatataatcaaagc-3′ SEQ ID primer:NO.: 42 Exon 18 Sense  5′-cacacagctgatgaatgtgc-3′ SEQ ID amplificationprimer: NO.: 43 Antisense 5′-tgaagggctacactttctgg-3′ SEQ ID primer:NO.: 44 Exon 19 Sense  5′-tctgccctcctattccaatg-3′ SEQ ID amplificationprimer: NO.: 45 Antisense 5′-gcccttgtcttccagaaatg-3′ SEQ ID primer:NO.: 46 Exon 20 Sense  5′-aaaaattacatcctttacatcaaactg-3′ SEQ IDamplification primer: NO.: 47 Antisense 5′-ttttgcatgcatagattttcc-3′SEQ ID primer: NO.: 48 Exon 21 Sense  5′-tgaaccttgcttttacatatcc-3′SEQ ID amplification primer: NO.: 49 Antisense5′-acccatctgggctcataaac-3′ SEQ ID primer: NO.: 50 Exon 22 Sense 5′-tgtcttggtccaaaatctgtg-3′ SEQ ID amplification primer: NO.: 51Antisense 5′-ttggtcgtttatgctttattcg-3′ SEQ ID primer: NO.: 52 Exon 23Sense  5′-ccctaaaggccaatttcagg-3′ SEQ ID amplification primer: NO.: 53Antisense 5′-atttggcagagaaaacactcc-3′ SEQ ID primer: NO.: 54 Exon 24Sense 5′-gagatttgggggtgtttgtc-3′ SEQ ID amplification primer:  NO.: 55Antisense 5′-ggattgtaatggggtgcttc-3′ SEQ ID primer: NO.: 56 Exon 25Sense  5′-caaaaatcagggccaatgac-3′ SEQ ID amplification primer: NO.: 57Antisense 5′-tgattgctgggatgatcttg-3′ SEQ ID primer: NO.: 58 Exon 26(1)Sense  5′-aggactctgaaccttaccttgg-3′ SEQ ID amplification primer: NO.: 59Antisense 5′-ccatgaatcgctcttccatc-3′ SEQ ID primer: NO.: 60 Exon 26(2)Sense  5′-tgtgggaacccatctgttg-3′ SEQ ID amplification primer: NO.: 61Antisense 5′-gtttgctgacaaggggtcac-3′ SEQ ID primer: NO.: 62

Tables 13 and 14 show nucleotide sequences of primer pairs used fordetecting the CACNA1A gene genome. In Tables 13 and 14, for example, E1Findicates an Exon 1 amplification sense primer, and E1Rv indicates anExon 1 amplification antisense primer.

TABLE 13  Exon 1 CACNA1A-E1F: 5′-tctccgcagtcgtagctccag-3′ SEQ ID NO.: 63amplification CACNA1A-E1Rv: 5′-agagattctttcacactcctcc-3′ SEQ ID NO.: 64Exon 2 CACNA1A-E2F: 5′-tttagaagtcacctgatctggg-3′ SEQ ID NO.: 65amplification CACNA1A-E2Rv: 5′-gacagagcgagactctggttca-3′ SEQ ID NO.: 66Exon 3 CACNA1A-E3F: 5′-gacaagagaactctgcaagagg-3′ SEQ ID NO.: 67amplification CACNA1A-E3Rv: 5′-atacagctgagacatggaggtg-3′ SEQ ID NO.: 68Exon 4 CACNA1A-E4F: 5′-tttatcccgtgaggcaggtactg-3′ SEQ ID NO.: 69amplification CACNA1A-E4Rv: 5′-cctcctgagatgctctgcatag-3′ SEQ ID NO.: 70Exon 5 CACNA1A-E5F: 5′-tgtggtgcttccttcaccattg-3′ SEQ ID NO.: 71amplification CACNA1A-E5Rv: 5′-cagaggctatttcactcactgc-3′ SEQ ID NO.: 72Exon 6 CACNA1A-E6F: 5′-ccccaaagccaaacattgatctc-3′ SEQ ID NO.: 73amplification CACNA1A-E6Rv: 5′-actctgattgtccacacacactg-3′ SEQ ID NO.: 74Exon 7 CACNA1A-E7F: 5′-cagaaaacgttcctccatttccc-3′ SEQ ID NO.: 75amplification CACNA1A-E7Rv: 5′-aagcttcaatggcctctacttgg-3′ SEQ ID NO.: 76Exon 8 CACNA1A-E8F: 5′-gccatactctggcttttctatgc-3′ SEQ ID NO.: 77amplification CACNA1A-E8Rv: 5′-cgtgatgtcagatcctggcttc-3′ SEQ ID NO.: 78Exon 9 CACNA1A-E9F: 5′-gttggctattgctactgttgcg-3′ SEQ ID NO.: 79amplification CACNA1A-E9Rv: 5′-gatccttagaaccagtcacctg-3′ SEQ ID NO.: 80Exon 10 CACNA1A-E1OF: 5′-tgatagtgccaccttgaacctc-3′ SEQ ID NO.: 81amplification CACNA1A-E1ORv: 5′-tgatgtaatctgcccaggacac-3′ SEQ ID NO.: 82Exon 11 CACNA1A-E11F: 5′-ctgcaacagagaactatcagcc-3′ SEQ ID NO.: 83amplification CACNA1A-E11Rv: 5′-aagagaagtggaaaaagggtgtg-3′SEQ ID NO.: 84 Exon 12 CACNA1A-E12F: 5′-gtagttctagcatgttggaggc-3′SEQ ID NO.: 85 amplification CACNA1A-E12Rv: 5′-atctgtcattccaggcaagagc-3′SEQ ID NO.: 86 Exon 13~15 CACNA1A-E13F: 5′-atggatgaatgagggggtcaag-3′SEQ ID NO.: 87 amplification CACNA1A-E15Rv: 5′-agcaggcactttcatctgtgac-3′SEQ ID NO.: 88 Exon 13~15 CACNA1A-E13F2: 5′-tccatttggagggaggagtttg-3′SEQ ID NO.: 89 amplification CACNA1A-E15Rv: 5′-agcaggcactttcatctgtgac-3′SEQ ID NO.: 88 Exon 14~15 CACNA1A-E14F: 5′-cctccagaaagttgggaaagtg-3′SEQ ID NO.: 90 amplification CACNA1A-E15Rv: 5′-agcaggcactttcatctgtgac-3′SEQ ID NO.: 88 Exon 16~17 CACNA1A-E16F: 5′-aaggagaagccaacacggagtc-3′SEQ ID NO.: 91 amplification CACNA1A-E17Rv:5′-ggtggtaactttgccagagaaac-3′ SEQ ID NO.: 92 Exon 18 CACNA1A-E18F:5′-agcaggtacccattccaattgg-3′ SEQ ID NO.: 93 amplification CACNA1A-E18Rv:5′-aatctgtgcctgggatagtgtg-3′ SEQ ID NO.: 94 Exon 19 CACNA1A-E19F:5′-cctgactcagatgctcacagac-3′ SEQ ID NO.: 95 amplification CACNA1A-E19Rv:5′-acacagcacgtgctactttggc-3′ SEQ ID NO.: 96 (1) Exon 19 CACNA1A-E19F2:5′-gaggacttcctcaggaaacag-3′ SEQ ID NO.: 97 amplification CACNA1A-E19Rv:5′-acacagcacgtgctactttggc-3′ SEQ ID NO.: 96 (2) Exon 20 CACNA1A-E20F:5′-agatggaatcttagctaggatcc-3′ SEQ ID NO.: 98 amplificationCACNA1A-E20Rv: 5′-aattatctcactgaaccctccac-3′ SEQ ID NO.: 99 Exon 21CACNA1A-E21F: 5′-agaaatgtcagccgcttcttgc-3′ SEQ ID NO.: 100 amplificationCACNA1A-E21Rv: 5′-ggtggtcaacactcactcattg-3′ SEQ ID NO.: 101 Exon 22CACNA1A-E22F: 5′-tttgttgtgtaggaggccttgg-3′ SEQ ID NO.: 102 amplificationCACNA1A-E22Rv: 5′-aacatcccaccctacctatgag-3′ SEQ ID NO.: 103

TABLE 14  Exon 23 CACNA1A-E23F: 5′-cctgcgcaactgtatatagcag-3′SEQ ID NO.: 104 amplification CACNA1A-E23Rv:5′-ctcaacctcctgatctcaagtg-3′ SEQ ID NO.: 105 Exon 24 CACNA1A-E24F:5′-cccaaagtttggatctaagagcc-3′ SEQ ID NO.: 106 amplificationCACNA1A-E24Rv: 5′-aaagccatcgaagctcttcctg-3′ SEQ ID NO.: 107 Exon 25CACNA1A-E25F: 5′-caggtgaaatggaccactcttc-3′ SEQ ID NO.: 108 amplificationCACNA1A-E25Rv: 5′-tccttgagcagtgtacaacctg-3′ SEQ ID NO.: 109 Exon 26CACNA1A-E26F: 5′-gaatgccaggattgagtccaac-3′ SEQ ID NO.: 110 amplificationCACNA1A-E26Rv: 5′-gaatgtgctggaaagtggagac-3′ SEQ ID NO.: 111 Exon 27CACNA1A-E27F: 5′-cactgcttcccaagcagtctag-3′ SEQ ID NO.: 112 amplificationCACNA1A-E27Rv: 5′-attacaggcgtgagccaccatg-3′ SEQ ID NO.: 113 Exon 28CACNA1A-E28F: 5′-tttccctctgttcctgttctgc-3′ SEQ ID NO.: 114 amplificationCACNA1A-E28Rv: 5′-ttcggttgggacaatgcttctg-3′ SEQ ID NO.: 115 Exon 29CACNA1A-E29F: 5′-ctcaagcaactgtagctgttgg-3′ SEQ ID NO.: 116 amplificationCACNA1A-E29Rv: 5′-ttatcagggtagaggcaggaac-3′ SEQ ID NO.: 117 Exon 30CACNA1A-E30F: 5′-gtgaaaagaagagcctagtccg-3′ SEQ ID NO.: 118 amplificationCACNA1A-E30Rv: 5′-atggtaacactcacaggttggg-3′ SEQ ID NO.: 119 Exon 31CACNA1A-E31F: 5′-gcccttcgaacaaccataactg-3′ SEQ ID NO.: 120 amplificationCACNA1A-E31Rv: 5′-cctacagccaagctttggttac-3′ SEQ ID NO.: 121 Exon 32CACNA1A-E32F: 5′-cccattggttttttggcactgg-3′ SEQ ID NO.: 122 amplificationCACNA1A-E32Rv: 5′-ggacagacagacagaggagag-3′ SEQ ID NO.: 123 Exon 33~35CACNA1A-E33F: 5′-tgttggttggcttcatgtaggg-3′ SEQ ID NO.: 124 amplificationCACNA1A-E35Rv: 5′-cagaattatcagagcaggtccc-3′ SEQ ID NO.: 125 Exon 36CACNA1A-E36F: 5′-tctcagctcccagtaaaaggag-3′ SEQ ID NO.: 126 amplificationCACNA1A-E36Rv: 5′-caacagtgctgagtttgagacg-3′ SEQ ID NO.: 127 Exon 37CACNA1A-E37F: 5′-ggcctctgtgtacatgtctttg-3′ SEQ ID NO.: 128 amplificationCACNA1A-E37Rv: 5′-gggtatgcaagggtgatgattc-3′ SEQ ID NO.: 129 Exon 38CACNA1A-E38F: 5′-tgtttctccccacctctcttc-3′ SEQ ID NO.: 130 amplificationCACNA1A-E38Rv: 5′-aaaaaaacccagtgcctggacg-3′ SEQ ID NO.: 131 Exon 39CACNA1A-E39F: 5′-agaaactgagtactgggacagg-3′ SEQ ID NO.: 132 amplificationCACNA1A-E39Rv: 5′-ggaagagtgaatgaagatccgg-3′ SEQ ID NO.: 133 Exon 40~41CACNA1A-E40F: 5′-aaagattggggtctcgttctcg-3′ SEQ ID NO.: 134 amplificationCACNA1A-E41Rv: 5′-ccctcatattccagttggttcc-3′ SEQ ID NO.: 135 Exon 42~44CACNA1A-E42F: 5′-gtgtgtgtgtgtgtatactggg-3′ SEQ ID NO.: 136 amplificationCACNA1A-E44Rv: 5′-cagactgcttcagagactgaag-3′ SEQ ID NO.: 137 Exon 45CACNA1A-E45F: 5′-ccgatttctcttgatgccagtg-3′ SEQ ID NO.: 138 amplificationCACNA1A-E45Rv: 5′-agggtgcgattgccaaagaaag-3′ SEQ ID NO.: 139 Exon 46~47CACNA1A-E46F: 5′-acccagagccctgattgatcag-3′ SEQ ID NO.: 140 amplificationCACNA1A-E47Rv: 5′-ttggatggggtatccccttctc-3′ SEQ ID NO.: 141 Exon 48CACNA1A-E48F: 5′-tctcttcctcccaatcccgtg-3′ SEQ ID NO.: 142 amplificationCACNA1A-E48Rv: 5′-tgcccaggagggtctcttttg-3′ SEQ ID NO.: 143

INDUSTRIAL APPLICABILITY

As described above, by detecting the presence of a mutation on bothα-subunit type 1 of voltage-gated sodium ion channel Na_(V)1.1 andα-subunit type 1 of voltage-gated calcium ion channel Ca_(V)2.1, it ispossible to obtain data for assessing a potential for development ofDravet syndrome of a subject who has not yet been subjected to onset ofDravet syndrome, with high accuracy. Hence, it is possible todistinguish a patient of Dravet syndrome that requires treatment by aspecialist, out of benign febrile seizure patents, at an initial stageof disease under the age of one. Hence, it is possible to use not onlyin the field of diagnosis medical treatment such as medical devices,diagnosis kits and the like, but broadly in the health science andmedical field industry.

Moreover, in the present invention, by introducing a mutation on both ofα-subunit type 1 of voltage-gated sodium ion channel Na_(V)1.1 andα-subunit type 1 of voltage-gated calcium ion channel Ca_(V)2.1, it ispossible to produce a model animal of Dravet syndrome. Such a modelanimal of Dravet syndrome can be used for development of medicament andtreatment methods of Dravet syndrome. Hence, the present invention canbe widely used in the industry of life science fields including thepharmaceutical field.

1. A method of obtaining data for assessing potential for development ofDravet syndrome, the method comprising: with use of a sample taken froma subject, detecting whether or not a mutation exists on α-subunit type1 of voltage-gated sodium ion channel Na_(V)1.1; and detecting whetheror not a mutation is on α-subunit type 1 of voltage-gated calcium ionchannel Ca_(V)2.1.
 2. The method according to claim 1, wherein themutation on the α-subunit type 1 of the voltage-gated sodium ion channelNa_(V)1.1 is at least one of mutations recited in Table 1, and themutation on the α-subunit type 1 of the voltage-gated calcium ionchannel Ca_(V)2.1 is at least one of mutations recited in Table
 2. 3.The method according to claim 1, further comprising: detecting a changein activity of the voltage-gated sodium ion channel Na_(V)1.1; anddetecting a change in activity of the voltage-gated calcium ion channelCa_(V)2.1.
 4. A kit for assessing a potential for development of Dravetsyndrome, the kit comprising: a polynucleotide being used fordetermining a mutation on α-subunit type 1 of voltage-gated sodium ionchannel Na_(V)1.1; and a polynucleotide being used for determining amutation on α-subunit type 1 of voltage-gated calcium ion channelCa_(V)2.1.
 5. A model animal of Dravet syndrome, having a mutation onboth α-subunit type 1 of voltage-gated sodium ion channel Na_(V)1.1 andα-subunit type 1 of voltage-gated calcium ion channel Ca_(V)2.1.
 6. Amethod of producing a model animal of Dravet syndrome as set forth inclaim 5, the method comprising: introducing a mutation on a α-subunittype 1 of the voltage-gated sodium ion channel Na_(V)1.1; andintroducing a mutation on a α-subunit type 1 of the voltage-gatedcalcium ion channel Ca_(V)2.1.
 7. A cell, having a mutation on bothα-subunit type 1 of voltage-gated sodium ion channel Na_(V)1.1 andα-subunit type 1 of voltage-gated calcium ion channel Ca_(V)2.1.
 8. Amethod of producing a cell as set forth in claim 7, the methodcomprising: introducing a mutation on a α-subunit type 1 of thevoltage-gated sodium ion channel Na_(V)1.1; and introducing a mutationon a α-subunit type 1 of the voltage-gated calcium ion channelCa_(V)2.1.
 9. A screening method of a drug for treating Dravet syndrome,the method comprising: administering a candidate agent to the modelanimal of Dravet syndrome as set forth in claim 5; and assessing whetheror not the administering of the candidate agent has made Dravet syndromeimprove or cure in the model animal of Dravet syndrome.
 10. A screeningmethod of a drug for treating Dravet syndrome, the method comprising:administering a candidate agent to the cell as set forth in claim 7; andassessing whether or not the administering of the candidate agent hasmade activity of the voltage-gated sodium ion channel Na_(V)1.1 and/oractivity of the voltage-gated calcium ion channel Ca_(V)2.1 change inthe cell.
 11. The method according to claim 2, further comprising:detecting a change in activity of the voltage-gated sodium ion channelNa_(V)1.1; and detecting a change in activity of the voltage-gatedcalcium ion channel Ca_(V)2.1.